Serum and exosome WNT5a levels as biomarkers in non-small cell lung cancer

Abstract

Primary tumor tissue and serum samples were collected from patients with histologically confirmed NSCLC before therapy. Healthy serum donors served as controls. Exosomes were isolated, then exosome number and size were measured, and WNT5A protein levels were identified in tissue and in vesicle-free, vesicle-bound fractions of the serum. Elevated WNT5A levels on the serum-exosome surface correlated with distant metastasis, advanced disease stage, and lymph node involvement in LUSC but not in LUAD patients. Moreover, a high WNT5A exosome surface level was associated with a poor response to therapy in LUSC patients. Additionally, serum-exosome surface+cargo WNT5A content distinguished LUAD and LUSC subtypes. WNT5A, in its serum exosome-bound form, serve as a biomarker for differentiating NSCLC subtypes and predicting disease progression. The information is available from a simple serum sample at the time of diagnosis.

Description

[0001] Serum and Exosome WNT5A Levels as Biomarkers in Non-Small Cell Lung Cancer

[0002] FIELD OF THE INVENTION

[0003] This invention relates to the field of lung cancer. More particularly, this invention relates to methods of differentiating between, the subtypes of a form of lung cancer (LC). This invention further relates to determining a prognosis in at least one subtype of at least one form of lung cancer.

[0004] The present invention assessed the WNT5A protein as a potential biomarker for diagnosing patients and predicting prognosis to assist in therapy selection.

[0005] BACKGROUND

[0006] Despite significant advances in the treatment of lung cancer (LC), there are no reliable biomarkers to effectively predict therapy response and overall survival (O / S) in non-small cell lung cancer (NSCLC) subtypes. While targeted therapies have improved survival rates in lung adenocarcinoma (LUAD), effective treatment options for lung squamous cell carcinoma (LUSC) are still limited.

[0007] By 2020, LC became the second most common cancer and the leading cause of death worldwide [Thandra et al., 2021; Wang et al., 2022], NSCLC represents 85% of all LC cases, with LUAD comprising 50% and LUSC accounting for 30% [Perez-Moreno et al., 2012], Recent reports by the American Society of Clinical Oncology (ASCO) have shown significant progress in treating NSCLC [Mountzios et al., 2024], Nonetheless, the 5-year survival of LC is still lower than any other cancer type [Bade et al., 2020],

[0008] Many LUAD subtypes have well-described mutations that can be targeted with specific drugs that increase survival. The most well-known mutations that determine therapy in LUAD patients include KRAS, EGFR, and ALK mutations [Sahu et al., 2023], however, additional targets have been discovered by technologies like next-generation sequencing (NGS) [Imyanitov et al., 2020], Besides therapies based on the genetic background other cancer-related therapies may be applied.

[0009] In contrast to LUAD, LUSC is more strongly associated with cigarette smoking, resulting in a highly variable mutational background, often in combinations of gene amplifications, gene fusions, tumour suppressor mutations and point mutations[Sands et al., 2020], Therapeutic response to mutation-based therapies are variable [Saez-Ibanez et al., 2023] and complex approaches often required.

[0010] The answer potentially lies in the complexity of the carcinogenic process, where not just genetic mutations are responsible for the disease outcome but highly diverse and malfunctioning signalling pathways, including the WNT pathways [Pongracz et al., 2006], In the carcinogenic process, the transforming members of the WNT ligand family have been investigated in depth [Rapp et al., 2016], One of the non-transforming family members, WNT5A, has been identified in several studies as one of the primary regulators of a wide range of squamous cell carcinomas (SCCs), including oral SCC [Prgomet et al., 2017], oesophageal SCC [Feng et al., 2022a], cutaneous SCC [Sherwood et al., 2016], and lung SCC (LUSC) [Rapp et al., 2016; Yao et al., 2014; Bartis et al., 2013; Huang et al., 2005], however its role in processes connected to cancer progression, such as distant metastasis or lymph node involvement was not yet discussed in these studies.

[0011] In a 2017 study, Prgomet et al. [Prgomet et al., 2017] link a statistically significantly higher expression level of WNT5A to oral SCC, however they found no correlation between WNT5A levels and measured tumor size, making the present invention's usage of the protein as a prognostic marker surprising. Although WNT5A lacks transforming activity, it occasionally signals via the canonical or p-catenin-dependentpathway, which is the traditional signalling pathway for transforming canonical WNT ligands [Pongracz et al., 2006], The secreted WNT5A is primarily known as a noncanonical WNT ligand that contributes less to the initiation of carcinogenesis but strongly supports cancer progression [Feng et al., 2022b] by affecting cell migration, invasion, inflammation, and angiogenesis by diversely binding to the Frizzled (FZD), RYK, and ROR2 receptors [Asem et al., 2016], Comparative analysis of WNT pathways in primary LUAD and LUSC tumour tissues revealed increased mRNA levels of the noncanonical WNT5A in LUSC [Chen et al., 2022; Xue et al., 2023], In a recent clinical study in which the expression and clinical impact of the WNT5A, WNT7B, FZD7 and GPC1 proteins were investigated in tumour samples, LUSC patients with no or minimal increase in WNT5A protein levels had the longest survival [Chen et al., 2022],

[0012] However, no previous study or invention suggested that additional information can be gathered from a patient's exosomal WNT5A protein profile, regarding cancer diagnosis and prognosis. As most of the studies focused on WNT5A protein at the tumour tissue level, less information is available on circulating ligands and their effects. To address where WNT5A is located, WNT5A in tumour tissues were compared with extracellular, circulating WNT5A in patient sera, both as vesicle-bound and vesicle-free soluble molecules. The lipophile [He et al., 2023] WNT5A can be transported by several lipid-containing carrier structures, including the secreted WNT interacting protein, heparan sulphate proteoglycans (HSPGs), cytonemes and the most broadly studied members of the extracellular vesicles (EV), exosomes [Mehta et al., 2021], Previous research into the role of vesicle-free and vesicle-bound forms of WNT5A has shown that the ratio of secreted versus vesicle-bound WNT5A depends on the cell type and the cellular context [Huang et al., 2017; Shi et al., 2017] and apparently revealed varying concentrations of WNT5A in LUAD and LUSC in tumour tissue. While secreted WNT5A appears to promote a more aggressive cancer phenotype, WNT5A in extracellular vesicles may exhibit context-dependent effects, potentially inhibiting or promoting cancer progression [Wang et al., 2017; Zeng et al., 2016],

[0013] The prognostic, diagnostic or predictive role of serum exosome-bound and exosome-free soluble WNT5A in LUAD and LUSC patients has not been investigated yet.

[0014] Methods of differentiating between LUAD and LUSC by using serum markers is a challenge today. Cao B et al. [Cao et al. 2021] identify blood-based exosomal mRNA markers that can distinguish between the two main subtypes of NSCLC: Lung Adenocarcinoma (LUAD) and Lung Squamous Cell Carcinoma (LUSC). Exosomes examined were present in samples taken from the diagnosed patients' blood and compared to healthy controls. The authors found that TP63 and KRT5 were significantly higher in LUSC exosomes, whereas CEACAM6 and SFTPB were significantly higher in LUAD exosomes. A combination of all four genes achieved a relatively high diagnostic accuracy (AUC of 0.822) compared to any single gene.

[0015] The present inventors made efforts to reveal the role of WNT5A as a potential biomarker for clinical applications including disease progression, therapy response and patient survival [Bartis et al., 2013; Chandrashekar et al., 2022],

[0016] Wang, J. et al. [Wang et al. 2022] provided an extensive review on expression, prognosis and immune infiltration of all 19 members of the Wingless-type (WNT) gene family in NSCLC. The study utilized multiple bioinformatics databases (TIMER, UALCAN, TCGA) and validated results using 20 pairs of clinical NSCLC tissue samples and cell lines via qRT-PCR and Western blot. While WNT2B and WNT7A were identified as providing critical prognostic insights, it is mentioned that LUSC appeared to promote WNT5A expressionin cancer tissue. However, the present inventors observed that elevated levels of WNT5A in resected tumour tissue were not associated with diminished OS and did not differentiate NSCLC subtypes [Wang et al., 2022],

[0017] Xue, W. et al. [Xue. et al. 2023] review current literature on 19 WNT ligands, their signaling pathways (canonical vs. non-canonical), and the regulatory roles of non-coding RNAs. The authors conclude that most WNTs (e.g., WNT1, 2, 3) are oncogenic, promoting tumor growth via the canonical p-catenin pathway. The authors devote brief chapters to these ligands and characterize WNT5a as a para- and autocrine p-catenin-independent ligand that has been shown to inhibit or induce cancer. The authors conclude that in NSCLC patients with higher WNT5a expression levels had advanced TNM stages and poor outcomes. However, no prognostic and diagnostic use is hinted and in fact tumor biopsies which might be used for such purpose based on Xue et al. are of small utility.

[0018] These documents do not disclose or hint toward measurement of Wnt5a serum levels. Importantly, an altered level in the tumor does not mean the serum level would be parallel to any extent. It is not clearly understood why an increase in tumor takes place and this information cannot be use in serum diagnostics and it is particularly unclear whether a content of an exosome is related to tumor expression.

[0019] The methods described in current published patent applications in the field of the invention do not present solutions for the above discussed problems either. One patent application, WO2019094692A2 lists WNT5A as an exosomal biomarker of cancer and cancer progression but does not mention its potential usage in the differential diagnosis of NSCLC subtypes and it does not outline the utility of exosomal WNT5A in determining a prognosis in LUSC patients.

[0020] Another patent publication, AU2024202148A1 describes a method of determining NSCLC aggressiveness and progression based on the expression of a plurality of biomarkers in an exosome sample of a patient, but it does not mention WNT5A as one of the biomarkers or a method of differentiating between LUAD and LUSC. None of the current patents or applications found in public databases mention the use of the number of exosomes in a serum sample of an NSCLC patient for the differential diagnosis of NSCLC subtypes.

[0021] The present inventors unexpectedly recognized that identifying serum WNT5A levels may be indicative in subtypes of NSCLC. In particular, WNT5A proteins bound to exosomes was found to be a biomarker of NSCLC stages and prognosis, as well as treatment response.

[0022] In the present invention, high levels of WNT5A bound to exosomes were associated with LUSC patients and shorter overall survival (OS), while higher WNT5A in exosome-free serum predicted better survival associated with LUAD.

[0023] This allows an early discovery of NSCLC patient status and prognosis with a simple and convenient method based on serum samples.

[0024] BRIEF DESCRIPTION OF THE INVENTION

[0025] 1. The invention relates to a method for diagnosing subtypes or differentiating between subtypes in nonsmall cell lung cancer (NSCLC) patients, the method comprising:

[0026] - measuring the exosome concentration in a serum sample comprising exosomes obtained from / of an NSCLC patient, and- measuring the exosome-bound WNT5A level in serum-derived exosomes (preferably normalized levels), preferably the WNT5A content of the exosomes relative to the exosome concentration (WNT5A content per exosome (particle, small EV)) in a serum sample comprising exosomes obtained from / of an NSCLC patient, wherein preferably the WNT5A content of the exosomes relative to the exosome concentration is the normalized exosomal WNT5A level, wherein preferably WNT5A level is WNT5A protein level; wherein preferably the exosome concentration is measured (i) in the serum sample, and

[0027] - determining the NSCLC subtype for the patient based on the measured exosome-bound WNT5A level (WNT5A content / exosome level) of circulating exosomes, wherein if the (preferably normalized) exosome-bound WNT5A level elevated in comparison with a healthy control level (and preferably higher than a LUAD control level) the NSCLC is considered as lung squamous cell carcinoma (LUSC), and, preferably, if the (preferably normalized) exosome-bound WNT5A level is lower than a LUSC control level the NSCLC is considered as lung adenocarcinoma (LUAD).

[0028] Optionally, a serum sample or a sample from the serum sample from a patient is provided.

[0029] Preferably the exosome particle is a small extracellular vesicle (EV). Exosomes are small spheroid membrane compartments or EVs of 30-150 nm preferably having surface markers in particular CD9, CD81 es DC63. Preferably exosomes are identified by methods as disclose herein e.g. in the examples.

[0030] Optionally a therapy is proposed depending on the disease subtype of the patient or a therapy corresponding thereto. Preferred therapies are disclosed e.g. in the detailed description of the invention. Preferably

[0031] in case of a patient with LUAD, the patient is proposed to be treated with an anti-LUAD therapy, preferably the LUAD-related mutation is determined and an anti-cancer therapy directed to the mutation;

[0032] in case of a patient with LUSC, the patient is proposed to be treated with an anti-LUSC therapy, preferably surgery and / or a complex therapy comprising therapies selected from the group consisting of chemotherapy, radiation therapy, chemoradiation, antibody therapy, eg. anti PD-L1 therapy etc.;

[0033] A patient is a subject under medical supervision or care. In a broad sense medical supervision or care is to be understood broadly, i.e. any health-related activity relating to the subject.

[0034] The invention is also related to method for differentiating between lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) in a subject having non-small cell lung cancer (NSCLC), the method comprising:

[0035] wherein a serum sample obtained or originated from the subject is provided;

[0036] - measuring (i) an exosome concentration in the serum sample or in a serum-derived exosome fraction; - measuring (ii) a WNT5A protein amount associated with serum-derived exosomes, wherein the WNT5A protein amount is a total exosomal WNT5A amount comprising WNT5A on an exosome surface and WNT5A in an exosome lumen (cargo);

[0037] - calculating (iii) a normalized exosomal WNT5A level as WNT5A amount per exosome particle based on the WNT5A protein amount of step (c) and the exosome concentration of step (b); and

[0038] - differentiating (iv) LUAD and LUSC based on the normalized exosomal WNT5A level, wherein a normalized exosomal WNT5A level above a predetermined threshold is indicative of LUSC, and a normalized exosomal WNT5A value at or below the predetermined threshold is indicative of LUAD. In a preferred option the exosome concentration is expressed as particles per unit volume.Thereby in the determining step the a differentiation between LUAD and LUSC is carried out.

[0039] In a preferred embodiment the exosome concentration is measured by nanoparticle tracking analysis (NTA) and expressed as particles / mL.

[0040] In a preferred embodiment WNT5A is measured by an antibody-binding assay, preferably an enzyme-linked immunosorbent assay (ELISA).

[0041] 2. Preferably, in the determining step

[0042] the NSCLC is considered as LUSC if the elevated exosome-bound WNT5A level is higher than a cut-off value for WNT5A content per exosomes differentiating between LUSC and LUAD, and preferably,

[0043] the NSCLC is considered as LUAD if the elevated exosome-bound WNT5A level is not higher (preferably lower) than a cut-off value for WNT5A content per exosomes differentiating between LUSC and LUAD.

[0044] 3. Preferably, in the determining step or in the differentiating step

[0045] the cut-off value for WNT5A content per exosomes (normalized exosomal WNT5A level) differentiating between LUSC and LUAD is between 2-3 x 10-9of picograms WNT5A per exosome (nanoparticle), preferably between 2,2-2,8 x 10-9of picograms WNT5A per exosome (nanoparticle), more preferably between 2,4-2,6 x 10-9of picograms WNT5A per exosome (nanoparticle).

[0046] Preferably, the cut-off value is a predetermined threshold.

[0047] Preferably, the predetermined threshold for the normalized exosomal WNT5A level is between 2×10-9and 3×10-9pg WNT5A per exosome particle.

[0048] Preferably, the predetermined threshold for the exosome-surface WNT5A normalized value is about 1.8×10-9pg WNT5A per exosome particle

[0049] Preferably, in the determining step

[0050] the NSCLC is considered as LUSC if the elevated exosome-bound WNT5A level is higher than a cut-off value for WNT5A content per exosomes differentiating between LUSC and LUAD, and preferably,

[0051] the NSCLC is considered as LUAD if the elevated exosome-bound WNT5A level is not higher (preferably lower) than a cut-off value for WNT5A content per exosomes differentiating between LUSC and LUAD.

[0052] 4. In a preferred embodiment

[0053] the exosome-bound WNT5A level in circulating exosomes is or comprises the WNT5A level on the surface of the exosomes.

[0054] In a preferred embodiment an increased (elevated) exosome surface WNT5A level is significantly elevated in comparison to a normal level (e.g. average or mean) of WNT5A level on the surface of the exosomes in healthy controls (HC) e.g. a cohort of HC or a normal level derived therefrom.

[0055] 5. Preferaby, measuring the total exosomal WNT5A amount comprises lysing the serum-derived exosomes and measuring WNT5A in the lysate.

[0056] More preferably, measuring the WNT5A protein amount associated with serum-derived exosomes comprises measuring a surface-associated WNT5A amount on intact exosomes without lysis.

[0057] 6. Preferably, the WNT5A level on the surface of the exosomes is measured by methods as described in the examples.

[0058] Preferably, the WNT5A level on the surface of the exosomes is measured by an antibody binding based method to measure the WNT5A amount or level and a method to assess the number or concentration, preferably number of exosomes, wherein preferably the exosomes are intact.In a preferred embodiment the WNT5A amount is measured by a protein assay e.g. an immunological assay like ELISA.

[0059] In a preferred embodiment the number of exosomes is measured by NTA (Nanoparticle tracking analysis). Characterization of exosomes is preferably carried out by TEM (Transmission electron microscopy plus preferably by EV Antibody array. In characterization of exosomes art guidelines can be followed. E.g exosomes can be 30-150 nm diameter spheroids preferably having CD9, CD81 es DC63 surface markers.

[0060] 7. Preferably, the WNT5A level in the lumen of the exosomes (cargo) is measured by methods as described in the examples.

[0061] Preferably, the WNT5A level on the lumen of the exosomes is measured by an antibody binding based method to measure the WNT5A amount or level and a method to assess the number or concentration, preferably number of exosomes, once the exosomes are disrupted.

[0062] In a preferred embodiment the WNT5A amount is measured by a protein assay e.g. an immunological assay like ELISA.

[0063] In a preferred embodiment the number of exosomes is measured by NTA (Nanoparticle tracking analysis). Characterization of exosomes is preferably carried out by TEM (Transmission electron microscopy plus preferably by EV Antibody array. In characterization of exosomes art guidelines can be followed. E.g exosomes can be 30-150 nm diameter spheroids preferably having CD9, CD81 es DC63 surface markers.

[0064] 8. Particularly preferably the method also comprises

[0065] - predicting disease progression, preferably by the expectable survival time of an NSCLC patient based on the exosome-bound WNT5A level, wherein an elevated exosome-bound WNT5A level indicates a shorter expectable survival time and a decreased exosome-bound WNT5A level indicates longer expectable survival time.

[0066] 9. Preferably, the method further comprising

[0067] - determining disease progression, preferably metastasis level and lymph node involvement, of an NSCLC patient having the subtype LUSC, based on the level of exosome-surface bound WNT5A protein measured on the circulating exosomes obtained from a serum sample of the patient, wherein, in an embodiment, an elevated exosome-surface bound WNT5A protein level compared to the normal (e.g. average or median) level among NSCLC patients having the subtype LUSC indicates a more progressed disease with a higher chance of distant metastasis and lymph node involvement.

[0068] In a preferred embodiment an increased (elevated) exosome surface WNT5A level is significantly elevated in comparison to a normal level (e.g. average or mean) of WNT5A level on the surface of the exosomes in LUSC mO stage patients or e.g. a cohort of LUSC mO or SD stage patients, or a normal level derived therefrom.

[0069] Alternatively, the references are WNT5A levels on the surface of the exosomes in LUAD patients or e.g. a cohort of LUAD (mO or SD stage) patients, or a normal level derived therefrom.

[0070] 10. In a highly preferred embodiment, the invention relates to a prognostic method wherein

[0071] - elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) at the therapy baseline is indicative of distant metastasis in patients with LUSC and / or

[0072] - elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is indicative of advanced stage of the disease, and / or- elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is indicative of lymph node involvement, and / or

[0073] - elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is predictive of a lack of response to therapy in LUSC patients.

[0074] In a highly preferred embodiment, the invention relates to method for determining a risk of progression of the disease in a subject having lung squamous cell carcinoma (LUSC), the method comprising:

[0075] - providing a serum sample obtained from the subject at a therapy baseline;

[0076] - measuring an exosome concentration in the serum sample, and measuring a surface-associated WNT5A protein amount on serum-derived exosomes; and

[0077] - calculating the amount of exosome surface WNT5A per exosome particle as an exosome-surface WNT5A normalized level; and

[0078] - determining the risk of progression of the disease (or progressed disease) when the exosome-surface WNT5A normalized level is above a predetermined threshold, wherein progressed disease comprises one or more of distant metastasis, advanced disease stage, and lymph node involvement.

[0079] Preferably the elevated WNT5A / particle on the exosome surface (elevated normalized level of WNT5A on particle surfaces) at the therapy baseline is indicative of distant metastasis in patients with LUSC.

[0080] 11. In a further aspect the invention relates to a method for predicting disease progression in in non-small cell lung cancer (NSCLC) patients, the method comprising:

[0081] - measuring the exosome-bound WNT5A level in circulating exosomes, obtained from a serum sample of an NSCLC patient preferably the WNT5A content of the exosomes relative to the exosome concentration (WNT5A content / exosome (nanoparticle)) in a sample comprising exosomes obtained from / of an NSCLC patient);

[0082] - predicting disease progression, preferably by the expectable survival time of an NSCLC patient based on the exosome-bound WNT5A level, wherein an elevated exosome-bound WNT5A level indicates a short expectable survival time and a decreased exosome-bound WNT5A level indicates long expectable survival time.

[0083] Preferably, the progressed disease comprises distant metastasis.

[0084] Preferably, the progressed disease comprises advanced disease stage.

[0085] Preferably, the progressed disease comprises lymph node involvement.

[0086] 12. Preferably, an exosome-bound WNT5A level is considered as elevated when it is higher than a cut-off value for WNT5A content per exosomes, and an exosome-bound WNT5A level is considered as decreased when it is not higher (preferably lower) than a cut-off value for WNT5A content per exosomes.

[0087] Preferably, the normalized exosome-bound WNT5A level is the WNT5A content of the exosomes relative to the exosome concentration in the serum, optionally expressed as WNT5A content per exosomes, and the pre-determined threshold level is a cut-off value for differentiating between a short expectable survival time and a long expectable survival time, preferably the same as the exosome-bound WNT5A level differentiating between LUSC and LUAD,

[0088] wherein in the determining stepthe NSCLC patient is considered as having a short expectable survival time if the elevated exosome-bound WNT5A level is higher than the cut-off value for differentiating between a short expectable survival time and a long expectable survival time, and preferably,

[0089] the NSCLC patient is considered as having a short expectable survival time, if the elevated exosome-bound WNT5A level is not higher (preferably lower) than a cut-off value for the exosome-bound WNT5A level differentiating between LUSC and LUAD.

[0090] 13. Preferably in the determining step

[0091] the cut-off value for WNT5A content per exosomes differentiating between a short expectable survival time and a long expectable survival time is between 2-3 x IO-9picograms of WNT5A per exosome (nanoparticle), preferably between 2,2-2,8 x 10-9picograms of WNT5A per exosome (nanoparticle), more preferably between 2,4-2,6 x 10-9picograms of WNT5A per exosome (nanoparticle).

[0092] Preferably, the cut-off value is a predetermined threshold.

[0093] Preferably, the predetermined threshold for the normalized exosomal WNT5A level is between 2×10-9and 3×10-9pg WNT5A per exosome particle.

[0094] Preferably, the predetermined threshold for the exosome-surface WNT5A normalized value is about 1.8×10-9pg WNT5A per exosome particle.

[0095] Preferably, in the determining step

[0096] the NSCLC is considered as LUSC if the elevated exosome-bound WNT5A level is higher than a cut-off value for WNT5A content per exosomes differentiating between LUSC and LUAD, and preferably,

[0097] the NSCLC is considered as LUAD if the elevated exosome-bound WNT5A level is not higher (preferably lower) than a cut-off value for WNT5A content per exosomes differentiating between LUSC and LUAD.

[0098] 14. Particularly preferably

[0099] the exosome-bound WNT5A level in circulating exosomes comprises or is the WNT5A level on the surface of the exosomes.

[0100] 15. Particularly preferably

[0101] the exosome-bound WNT5A level in circulating exosomes comprises both

[0102] the WNT5A level on the surface of the exosomes and

[0103] the WNT5A level in the lumen of the exosomes (cargo).

[0104] 16. In a preferred embodiment the method further comprising

[0105] - determining disease progression, preferably metastasis level and lymph node involvement, of an NSCLC patient having the subtype LUSC, based on the level of exosome-surface bound WNT5A protein measured on the circulating exosomes obtained from a serum sample of the patient, wherein an elevated exosome-surface bound WNT5A protein level compared to the median level among NSCLC patients having the subtype LUSC indicates a more progressed disease with a higher chance of distant metastasis and lymph node involvement.

[0106] optionally a therapy corresponding to disease progression of the patient is proposed or carried out. 17. In a preferred embodiment the method further comprising

[0107] - predicting treatment response of an NSCLC patient having the subtype LUSC, based on the level of exosome-surface bound WNT5A protein measured on the circulating exosomes obtained from a serum sample of the patient, wherein a decreased level of exosome-surface bound WNT5A predicts a betterresponse to treatment and an elevated level of exosome-surface bound WNT5A protein predicts a worse response to treatment.

[0108] In an embodiment, the invention relates to a method for predicting treatment response and / or overall survival in a subject having lung squamous cell carcinoma (LUSC), the method comprising:

[0109] - providing a serum sample obtained from the subject at a therapy baseline;

[0110] - measuring an exosome concentration in the serum sample and measuring a surface-associated WNT5A protein amount on serum-derived exosomes;

[0111] - calculating an exosome-surface WNT5A normalized value as WNT5A amount per exosome particle; and - predicting a lack of response to therapy and / or a shorter overall survival when the exosome-surface WNT5A normalized value is above a predetermined threshold.

[0112] Particularly preferably

[0113] the exosome-bound WNT5A level in circulating exosomes comprises the WNT5A level on the surface of the exosomes.

[0114] Preferably, the exosome-surface WNT5A normalized value is used, optionally in combination with a clinical response classification, to distinguish (i) a LUAD patient with progressive disease from (ii) a LUSC patient having a lack of response to therapy.

[0115] 18. In a highly preferred embodiment, the invention relates to a prognostic method wherein

[0116] - elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) at the therapy baseline is indicative of distant metastasis in patients with LUSC and / or

[0117] - elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is indicative of advanced stage of the disease, and / or

[0118] - elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is indicative of lymph node involvement, and / or

[0119] - elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) predictive of a lack of response to therapy in LUSC patients.

[0120] In a preferred embodiment

[0121] WNT5A transported on the exosome surface is used as a marker to distinguish LUAD patients with PD from LUSC patients with a lack of therapy response.

[0122] Preferably, the exosome-surface WNT5A normalized value is used, optionally in combination with a clinical response classification, to distinguish (i) a LUAD patient with progressive disease from (ii) a LUSC patient having a lack of response to therapy.

[0123] Preferably, the elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is increased (elevated) in comparison with the normal (average or median) value typical of LUSC stable disease (SD) patients.

[0124] Preferably, the elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is increased (elevated) in comparison with the normal (average or median) value typical of LUSC patients.

[0125] 19. In a further aspect the invention relates to a method for diagnosing subtypes in non-small cell lung cancer (NSCLC) patients, the method comprising:

[0126] - measuring WNT5A level in a serum sample, preferably in exosome free serum of an NSCLC patient, and- differentiating the NSCLC subtype based on the measured WNT5A level, wherein an WNT5A serum level elevated in comparison with a healthy control serum level indicates lung adenocarcinoma (LUAD), and a lower WNT5A level indicates lung squamous cell carcinoma (LUSC).

[0127] 20. In a further aspect the invention relates to a method for diagnosing non-small cell lung cancer (NSCLC) in a subject, said method comprising

[0128] - measuring the level of WNT5A protein in a serum sample of a subject;

[0129] - determining whether the subject has NSCLC, wherein an elevated WNT5A protein level of the serum sample, in comparison with a healthy WNT5A protein level, is indicative of NSCLC in said patient.

[0130] Preferably, in the determining step, the subject is considered having NSCLC if the elevated WNT5A protein level of the serum sample is higher than a cut-off value for WNT5A level.

[0131] Preferably, in the determining step, the cut-off value for WNT5A level in the serum sample differentiating between NSCLC and healthy subjects is 400 to 1000 pg WNT5A protein per ml serum.

[0132] 21. In a preferred embodiment the invention relates to any method of paragraph 20, wherein the serum sample is

[0133] a total serum sample or

[0134] an exosome-free serum sample, and

[0135] as the WNT5A level in the serum sample is determined

[0136] the WNT5A level in total serum and / or

[0137] the WNT5A level in exosome free serum, respectively.

[0138] Preferably, the exosome-free serum WNT5A level is considered decreased when it is below about 740 pg / mL

[0139] Alternatively, the cut-off value is 400 to 1000 pg WNT5A protein per ml serum.

[0140] 22. Preferably

[0141] the exosome-bound WNT5A level in circulating exosomes comprises

[0142] the WNT5A level in total serum, and

[0143] the cut-off value is 500 to 800 pg, preferably 600 to 700 pg WNT5A protein per ml total serum. Preferably

[0144] the exosome-bound WNT5A level in circulating exosomes comprises

[0145] the WNT5A level in exosome free serum and

[0146] the cut-off value is 400 to 650 pg, preferably 450 to 550 pg WNT5A protein per ml exosome free serum.

[0147] 23. In further embodiment the method comprises

[0148] predicting the expectable survival time of an NSCLC patient based on the WNT5A protein level of the test sample prepared from a serum sample the patient, wherein an elevated exosome-free serum WNT5A level indicates a longer expectable survival time and a decreased exosome-free serum WNT5A level indicates shorter expectable survival time.

[0149] 24. Preferably,

[0150] the serum sample is

[0151] an exosome-free serum sample, and

[0152] the WNT5A level in exosome free serum is determined, andthe subject is considered having a long survival time if the WNT5A protein level of the serum sample is higher than a cut-off value for WNT5A level, and having a short survival time if the WNT5A protein level of the serum sample is not higher or lower than a cut-off value for WNT5A level,

[0153] wherein the cut-off value differentiating between high and low survival time is 600 to 1000 pg, preferably 700 to 900 pg or 700 to 800 pg WNT5A protein per ml exosome free serum.

[0154] 25. The invention also relates to a method of treatment or selecting a treatment for a subject having nonsmall cell lung cancer (NSCLC), comprising:

[0155] (a) obtaining a serum sample from the subject;

[0156] (b) determining, from the serum sample, an exosome-surface WNT5A normalized value as an amount of WNT5A per exosome particle, preferably according to any of claims 1 to 10 or 11 to 19 or 20 to 24; and (c) selecting a treatment for the subject based on the exosome-surface WNT5A normalized value, wherein an exosome-surface WNT5A normalized value above in accordance with the subtype of the disease as determined in any of claims 1 to 10 or the disease progression as determined in any of claims 11 of 19 or any of the status as determined in any of claims 20 of 26. For example if a predetermined threshold is indicative that the subject has lung squamous cell carcinoma (LUSC) with an increased likelihood of poor response to therapy and / or reduced overall survival, the treatment is carried out accordingly.(LUSC PD). Other disclosed variants are provide further options in the Detailed description of different treatments of LUSC and LUAD.

[0157] Preferably, the predetermined threshold is about 1.8×10-9pg WNT5A per exosome particle.

[0158] 27. In an embodiment the invention relates to a method of treating a subject having lung squamous cell carcinoma (LUSC), comprising: (a) determining an exosome-surface WNT5A normalized value from a serum sample obtained from the subject at a therapy baseline; (b) identifying the subject as being at increased risk of poor response to therapy when the exosome-surface WNT5A normalized value is above a predetermined threshold; and (c) administering to the subject a cancer therapy and / or adjusting a cancer therapy regimen based on the identification in step (b).

[0159] Preferably the predetermined threshold is about 1.8×10-9pg WNT5A per exosome particle.

[0160] 28. The method of claims 25 to 28, wherein the disease is LUSC PD and wherein adjusting the cancer therapy regimen comprises selecting or recommending a more intensive therapy regimen, a combination therapy regimen, and / or an earlier assessment of therapeutic response for the subject. Preferably, the cancer therapy comprises one or more of chemotherapy, immunotherapy, radiotherapy, and targeted therapy.

[0161] In an embodiment, an exosome-free serum WNT5A level below about 740 pg / mL is indicative of reduced overall survival.

[0162] 29. A method of treating a subject having NSCLC, comprising: (a) determining a normalized exosomal WNT5A value as a total exosomal WNT5A amount per exosome particle, wherein the total exosomal WNT5A amount comprises WNT5A on an exosome surface and WNT5A in an exosome lumen (cargo); (b) classifying the subject as having LUSC when the normalized exosomal WNT5A value is above a predetermined subtype threshold, or classifying the subject as having LUAD when the normalizedexosomal WNT5A value is at or below the predetermined subtype threshold; and (c) administering a therapy regimen based on the classification in step (b).

[0163] Preferably the predetermined subtype threshold is about 2.5×10-9pg WNT5A per exosome particle or a threshold as disclosed herein.

[0164] 30. A method for staging lung squamous cell carcinoma (LUSC) in a subject, comprising performing the method of claim 9 and classifying the subject as having (i) low-risk LUSC when the exosome-surface WNT5A normalized value is at or below a first threshold, or (ii) high-risk LUSC when the exosome-surface WNT5A normalized value is above the first threshold.

[0165] 31. A kit for performing the method of any of claims above, comprising: (i) a WNT5A-specific binding reagent; (ii) one or more calibrators or controls; and (iii) instructions for determining a risk of progressed disease in LUSC based on an exosome-surface WNT5A normalized value.

[0166] ABBREVIATIONS LC Lung cancer

[0167] NSCLC Non-small cell lung cancer

[0168] LUAD Lung Adenocarcinoma

[0169] LUSC Lung Squamous Cell Carcinoma

[0170] TKI Tyrosine kinase inhibitor

[0171] EGFR Epidermal growth factor receptor

[0172] KRAS Kirsten rat sarcoma viral oncogene homologue

[0173] EML4-ALK echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase BRAF B-Raf proto-oncogene serine / threonine kinase

[0174] MET hepatocyte growth factor receptor tyrosine kinase

[0175] ROS1 ROS proto-oncogene 1

[0176] RET rearranged during transfection proto-oncogene

[0177] NTRK neurotrophic tyrosine receptor kinase

[0178] HER2 human epidermal growth factor receptor 2

[0179] FGFR 1-3 fibroblast growth factor receptors 1-3

[0180] PDGFRA platelet-derived growth factor receptor alpha

[0181] PTEN phosphatase and tensin homologue

[0182] TP53 tumour protein p53

[0183] EPHA2 ephrin type A receptor 2

[0184] AKT1 alpha serine / threonine-protein kinase

[0185] PD progressive disease

[0186] CR complete response

[0187] PR partial response

[0188] SD stable disease

[0189] NP nanoparticle

[0190] NGS Next Generation Sequencing

[0191] CPTAC Clinical Proteomic Tumor Analysis ConsortiumDEFINITIONS

[0192] Patient - a subject receiving or registered to receive medical treatment, or who is or is intended to be under medical or veterinarian observation, supervision or diagnosis.

[0193] Disease progression - The increasing severity of a type of cancer over time, determined by qualitative parameters such as the presence of lymph node involvement or distant metastasis in a patient.

[0194] Diagnostic biomarker - A numerical biological parameter, such as the concentration of a protein in a serum sample of a patient, wherein the value of the parameter can be used to determine the type or subtype of a disease in the patient.

[0195] Prognostic biomarker - A numerical biological parameter, such as the concentration of a protein in a serum sample of a patient, wherein the value of the parameter can be used to determine the progression of a disease in a patient.

[0196] Exosome-bound - Residing on the surface or in the lumen of an exosome.

[0197] Progressive disease - Cancer in late stage of disease progression, when there is about or at least 20% increase in tumor size or new lesions.

[0198] Partial response is defined by an about or at leaset 30% decrease in tumor size.

[0199] Stable disease means that there is not enough shrinkage for PR, but there is not enough growth for PD either.

[0200] Complete response means that all target lesions disappear.

[0201] The singular forms "a", "an" and "the", or at least "a", "an", include plural reference unless the context clearly dictates otherwise.

[0202] The term "comprises" or "comprising" or "including" are to be construed here as having a non- exhaustive meaning and allow the addition or involvement of further features or method steps or components to anything which comprises the listed features or method steps or components. " Comprising" can be substituted by "including" if the practice of a given language variant so requires or can be limited to "consisting essentially of" if other members or components are not essential to reduce the invention to practice.

[0203] BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 - Patient selection and clinical parameters.

[0204] (A) Flow chart of patient selection in our prospective study; (B) Clinicopathologic parameters of NSCLC patients.

[0205] FIGURE 2 - Characterization of serum-derived exosomes (n=5 each sample group).

[0206] A-C: Size distribution of exosomes measured by NTA. D: Serum exosome concentration (NPs / mL) in HC, LUAD, and LUSC samples as detected with NTA. E: Exosomes isolated from NSCLC patients were observed under electron microscopy with 50-150 nm in diameter (bar=200 nm). F: Representative Exo-Check Exosome Antibody Array for detecting exosome markers (CD81, CD63, ALIX, EpCam, ANXA5, and TSG101) and assessing cellular contamination (GM130).

[0207] FIGURE 3 - WNT5A levels in various fractions of the serum.

[0208] A: The concentration of WNT5A protein in serum, supernatant, and exosomes. B: Distribution of WNT5A within exosomes in NSCLC and HC samples. Quantitative analysis of WNT5A levels in serum (C) andsupernatant (D) among HC, LUAD and LUSC patients. E: WNT5A levels pg / particle as exosome surface F: WNT5A levels pg / particle as exosome surface+cargo

[0209] FIGURE 4 - WNT5A levels associated with overall survival, disease stage and therapy response.

[0210] A: Kaplan-Meier O / S distributions stratified by supernatant WNT5A level (cut-off: 740 pg / mL) (p=0.0042, from log-rank test). B: Kaplan-Meier O / S distributions stratified by exosome surface+cargo WNT5A level (cut-off: 2.5×10-9pg WNT5A / NP) (p=0.028, from log-rank test). WNT5A on the surface of exosomes is elevated in the LUSC subtype and associated with distant metastasis C: Univariable Cox regression analysis D: Multivariable Cox regression analysis ROC analysis was performed to calculate sensitivities and specificities for differentiation between LUAD and LUSC metastatic patients. AUC represents the diagnostic capacity.

[0211] (E) WNT5A on the surface of exosomes is elevated in the LUSC subtype and is associated with distant metastasis, (F) advanced disease stage, (G) positive lymph node status and (H) a lack of response to therapy. H: WNT5A transported on the exosome surface is a suitable marker in distinguishing LUAD patients with PD from LUSC patients with a lack of therapy response. (PR, partial response; PD, progressive disease; SD, stable disease).

[0212] (PD, progressive disease; PR, partial response; SD, stable disease).

[0213] FIGURE 5 - Schematic diagram of the predictive, prognostic and diagnostic role of peripheral WNT5A in LUAD and LUSC patients.

[0214] The cut-off values are summarized for each application. (Further validation is needed to confirm that the cut-off values are not data-set dependent).

[0215] FIGURE 6 - Schematic summary diagram of WNT5A exosome characteristics in sera of LUAD and LUSC patients.

[0216] FIGURE 7 - WNT5A protein in NSCLC solid tumor tissues and overall survival (O / S).

[0217] Representative images of WNT5A protein expression detected with immunohistochemical staining in LUAD (A) and LUSC (B) specimens (magnification 10X); (C) Independent dataset analysis (D) WNT5A was scored via a semiquantitative method based on the H-score; and (E) O / S of NSCLC patients.

[0218] FIGURE 8 - IPA analysis

[0219] IPA analysis of LUAD (A) and LUSC (B) exosome protein cargo demonstrating LUSC pathways complexity. DETAILED DESCRIPTION OF THE INVENTION WNT5A has a consistent association with various cancer types [Feng et al., 2022b], yet its potential as a biomarker has remained unexplored. The present inventors have separated vesicle-free and vesiclebound fractions of the serum and found that they have reliably different WNT5A levels and yield predictive, prognostic or diagnostic value for NSCLC.

[0220] The present invention is based on the surprising discovery that the levels of WNT5A protein, measured in distinct fractions of a serum sample collected from a NSCLC patient, such as exosome-free serum WNT5A level or serum-exosome-bound WNT5A level are accurate diagnostic and prognostic biomarkers of nonsmall cell lung cancer or at least one subtype of non-small cell lung cancer.

[0221] Thus, WNT5A, particularly its serum exosome-bound form, serves as a valuable biomarker for differentiating NSCLC subtypes and predicting disease progression. Importantly, the method can provide result from a simple serum sample at the time of diagnosis.In their present work the inventors collected primary tumor tissue and serum samples from a cohort of 60 patients with histologically confirmed NSCLC before therapy. Healthy serum donors served as controls. Exosomes were isolated, then exosome number and size were measured, and WNT5A protein levels were identified in tissue and in vesicle-free, vesicle-bound fractions of the serum by ELISA.

[0222] Surprisingly, extensive statistical analysis revealed that elevated WNT5A levels on the serum-exosome surface correlated with distant metastasis, advanced disease stage, and lymph node involvement in LUSC but not in LUAD patients. Moreover, a high WNT5A exosome surface expression was associated with a poor response to therapy and shorter O / S in LUSC patients. Additionally, serum-exosome surface + cargo WNT5A content distinguished LUAD and LUSC subtypes.

[0223] Initially, the inventors observed that elevated levels of WNT5A in resected tumour tissue were not associated with diminished OS and did not differentiate NSCLC subtypes [Wang et al., 2022], However, significant differences were detected in WNT5A levels between the LUAD and LUSC subtypes by separating the exosome-free serum and various exosome fractions. LUSC patients had lower levels of circulating exosomes compared to LUAD patients or HCs. Moreover, LUSC exosomes contained significantly more WNT5A both on their surface and inside as cargo, in comparison to LUAD patients. Additionally, the exosome-free serum of LUAD patients had notably higher levels of WNT5A than LUSC patients, indicating that the two subtypes of NSCLC have distinct extracellular WNT5A profiles suggesting that the location of WNT5A may play a significant role in the clinical outcome.

[0224] In one aspect, the invention provides a method of diagnosing the subtype of non-small cell lung cancer (NSCLC) in a patient, said method comprising the steps of acquiring a serum sample from the patient, measuring the exosome content of the serum sample, measuring the level of WNT5A protein in the serum sample, measuring the level of WNT5A protein in the exosome-free serum sample, calculating the level of exosome-bound WNT5A protein, based on the results of the measurements conducted on the exosome-free serum sample and diagnosing the subtype of NSCLC in the patient based on the results at least one of the preceding measurements or calculation.

[0225] In another related aspect, the invention provides a method of determining a prognosis for an NSCLC patient, having the subtype lung squamous-cell carcinoma (LUSC), said method comprising measuring the level of WNT5A protein bound to the surface of exosomes, isolated from a serum sample of the LUSC patient and determining disease progression e.g. the probability of metastasis and lymph node involvement in the patient.

[0226] In the present invention, high levels of WNT5A bound to exosomes were associated with LUSC patients and shorter OS, while higher WNT5A in exosome-free serum predicted better survival associated with LUAD, suggesting that the context of WNT5A presentation and mode of secretion is crucial for its function in cancer [Zhan et al., 2017; Zhang et al., 2017], In exploring the reasons for reduced overall survival in LUSC, we reviewed the literature and found that not only do the exosome numbers and WNT5A levels differ (Figure 6), but various other cargo molecules do as well. While LUSC exosomes also contain TP63 and KRT5 (Keratin5) mRNA, LUAD exosomes carry CEACAM6 (carcinoembryonic antigen cell adhesion molecule 6) and SFTB (surfactant protein B) mRNAs [Cao et al., 2021], TP63 is characteristic in squamous cell carcinomas [Yi et al., 2020] and, if TP63 protein is combined with SOX2 it is known to promote LUSC progression and serve as super-enhancers in squamous tumors [Liu et al., 2021], Our WNT5A data, alongwith existing literature, were analyzed using Ingenuity Pathway Analysis (IPA, Qiagen), revealing the complexity of the affected signaling pathways (Figure 8). This analysis indicated a significantly higher complexity in LUSC. We also theorized that the membrane protein WNT5A can facilitate the delivery of exosomes to target sites in both subtypes of NSCLC. This is particularly relevant because the common metastatic sites for both subtypes including the lung, bone, liver, and brain tissue, have receptors for WNT5A, such as ROR2 and FZDs [Kamizaki et al., 2024; DeBruine et al., 2017],

[0227] WNT5A signaling induces a Ca2+-dependent release of exosomes containing the immunomodulatory and pro-angiogenic proteins in melanoma cells; Exosome-release of these factors enhance the immunosuppressive and angiogenic capacity of the tumors thus rendering them more aggressive and more prone to metastasize. [Ekstrom et al., 2014], Significantly, a simple blood sample and measuring WNT5A in serum and exosome fractions at the time of diagnosis can identify advanced disease stage, lymph node involvement, distant metastasis, predict lack of therapy response, and reduced OS in LUSC. In LUAD the exosome-associated WNT5A levels were significantly lower and associated with higher OS time. The difference in location of serum WNT5A is an additional diagnostic biomarker which differentiates LUSC from LUAD. Contrary to previous studies[Lu et al., 2015], the present invention is the first one to identify the precise location and diagnostic and prognostic value of serum and serum-derived exosome-associated WNT5A with specified cut-off values (Figure 5). Although smaller sample availability leads to reduced statistical power, the results are promising and warrant further validation in a larger clinical cohort to confirm the clinical utility of the extracellular WNT5A as a biomarker.

[0228] In most cases there is a difference in therapeutic approaches of LUAD and LUSC patients.

[0229] Many LUAD subtypes have well-described mutations that can be targeted with specific drugs that increase survival. The most well-known mutations that determine therapy in LUAD patients include KRAS, EGFR, and ALK mutations [Sahu et al., 2023], In addition to the traditionally tested therapy targets, nextgeneration sequencing (NGS) identified mutations in BRAF, MET, ROS1, RET, NTRK1 / 2 / 3, and ERBB2 that offer additional therapy targets [Imyanitov et al., 2020], Thus LUAD patients may receive therapy either based on the genetic background of their tumours or other cancer-related therapies.

[0230] In contrast to LUAD, LUSC is more strongly associated with cigarette smoking, resulting in a highly variable mutational background, often in combinations of gene amplifications (CCND1 / 2 / 3, CDK4, FGFR1 / 2 / 3, MET, PDGFRA, PIK3CA, SOX2), gene fusions (FGFR3:: TACC3), tumour suppressor mutations (PTEN, TP53) and point mutations (EPHA2, AKT1, DDR2) and alterations in PIK3CA, FGFR1, and DDR2; i.e events in a variety of genes [Sands et al., 2020], Therefore, to date, mutation-based targeted therapy is not available for LUSC patients. Although immune checkpoint inhibitors are used to treat patients with LUAD and LUSC, the therapeutic response remains variable [Saez-Ibanez et al., 2023],

[0231] These genetic differences make it advantagous to tailor treatment strategies based on specific molecular profiles.

[0232] However, the differences between LUAD and LUSC extends beyond genetics, affecting targeted therapies, chemotherapy radiotherapy and immunotherapy regimens - and outcomes.

[0233] Chemotherapy, like pemetrexed-based chemotherapy often effective in LUAD patients but lacks substantial benefits in LUSC which reflects differences in thymidylate synthase expression. EGFR tyrosinekinase inhibitors (TKIs) were found to be effective in LUAD but not in LUSC. Unfortunately, absence of widespread targetable mutations in LUSC presents ongoing challenges.

[0234] Nevertheless, as precision medicine advances, specific treatments for LUAD and LUSC are evolving. A comprehensive understanding of the driver genes for LUAD and LUSC, now available, largely help the clinical practitioners to find appropriate targeted therapies. Modern drugs targeting the significantly expressed gene targets or signaling pathways have started to allow tailored treatment of LUAD and LUSC. Importantly, differences exist between LUAD and LUSC in their immune treatment microenvironment characteristics. The tumour microenvironment also varies significantly between LUAD and LUSC, impacting responses to immune checkpoint inhibitors. Anyway, the distinct driver genes associated with LUAD and LUSC can result in varying prognosis outcomes during clinical targeted therapy. Shen et al. [Shen et al. 2024] Review differences between lung adenocarcinoma and lung squamous cell carcinoma: Driver genes, therapeutic targets, and clinical efficacy which can be utilized in differential treatment of these subtypes.

[0235] In the present invention, optionally a therapy is proposed depending on the disease subtype of the patient or a therapy corresponding thereto. Preferred therapies are disclosed herein as examples used by skilled persons of the field.

[0236] Preferably, in case of a patient with LUAD, the patient is proposed to be treated with an anti-LUAD therapy, preferably the LUAD-related mutation is determined and an anti-cancer therapy directed to the mutation.

[0237] In case of a patient with LUSC, the patient is proposed to be treated with an anti-LUSC therapy, preferably surgery and / or a complex therapy comprising therapies selected from the group consisting of chemotherapy, radiation therapy, chemoradiation, antibody therapy, eg. anti PD-L1 therapy etc.

[0238] In summary, measuring specific extracellular forms of WNT5A in liquid biopsies can provide important clinical information that complements tissue-based biomarkers and supports personalised clinical decision-making. The current data has significant limitations and represents only the first step in biomarker discovery. Independent validation is essential but challenging at this time due to the absence of serum exosome protein databases. Further studies and statistical power analyses are needed to confirm our findings.

[0239] EXAMPLES EXAMPE1 - Databases and WNT5A in tumours

[0240] Database analysis

[0241] Independent data set was analysed for WNT5A protein from using the Human Protein Atlas CPTAC_dataset [Digre A et al., 2023], to support protein levels of WNT5A in LUAD and LUSC tumour tissues for validation of tissue WNT5A levels in the study cohort [Chandrashekar et al., 2022; Chandrashekar et al., 2017], UALCAN is a comprehensive and interactive web resource designed to identify biomarkers or perform in silico validation of potential genes of interest. Extensive information has been collected on WNT5A in the above database, including differential levels of WNT5A mRNA and protein in association with TNM status,stage, race, sex, smoking, etc., and overall survival [Chandrashekar et al., 2022; Chandrashekar et al., 2017],

[0242] In a previous study [Bartis et al., 2013] and data from the UALCAN database [Chandrashekar et al., 2022] comparing over 500 hundred resected tumour tissues and normal controls, it was found that WNT5A mRNA levels were significantly higher in LUSC tumour tissues compared to those in LUAD or normal control (p=l.llE-16). In contrast, the WNT5A mRNA level was not significantly different in LUAD tissues compared to normal controls (p=0.138). However, in contrast to mRNA, the level of WNT5A protein was almost the same in LUAD and LUSC tumour tissues (according to UALCAN). A significant increase in protein was only detected when compared with normal controls in both tumour types (LUAD and normal tissues, p=5.78E-10; LUSC and normal tissues, p= 5.26E-24).

[0243] WNT5A protein in the tumour tissue

[0244] In our operable patient cohort, we did not detect significant differences in WNT5A protein levels in LUAD and LUSC tumour tissues (Figure 7A-B). Analysis of independent protein CPTAC datasets supported these results. (Figure 7D). Additionally, our Kaplan-Meier analysis revealed no correlation between tumour tissue WNT5A levels and OS (logrank p=0.14, HR=0.41, 95% Cl 0.12-1.39) (Figure 7D). In line with previous research, our study demonstrated that LUAD patients survived significantly longer than LUSC patients after diagnosis (p = 0.0193) (Figure 7D-E).

[0245] EXAMPLE 2 - Patients

[0246] Sixty patients enrolled in the prospective study at the Department of Pulmonology, 1stDivision Internal Medicine, Clinical Centre, University of Pecs (Pecs, Hungary) between 01.02.2018 and 15.12.2018. Each participant signed an informed consent form and blood serum samples were collected before any treatment began. After the diagnosis of pulmonary carcinoma, rule-based therapy and data collection followed. Where surgery was possible, tissue samples were also collected and stored at the Department of Pathology, Clinical Centre, University of Pecs (Pecs, Hungary). The inclusion criteria were as follows: (i) histologically confirmed NSCLC stage l-IV (LUAD or LUSC) based on American Joint Committee on Cancer (AJCC, version 8) staging; (ii) no history of radiotherapy, immunotherapy, chemotherapy, or other treatments before diagnosis; (iii) availability of complete follow-up data including best overall response (BOR) and overall survival (OS); and (iv) availability of sufficient quantity and quality of serum samples. The exclusion criteria were as follows: (i) pathologically different diagnoses of NSCLC; (ii) patients with a history of a second primary malignancy; and (iii) serum samples or any other reason for the failure of quality control at any stage of the study. BOR was determined according to RECIST version 1.1 [Therasse et al., 2000], The time from the start of treatment to the end of any cause or the last follow-up date was defined as OS. Serum from healthy blood donors (HC) selected by a comparable age distribution to the study population were used as controls. The study was declared complete three years later, in 2021. The study was approved by the local Research Ethics Committee, University of Pecs (PTE_KK_RIKEB_6444 / 2016) and was conducted in compliance with the Declaration of Helsinki.

[0247] The study was designed following the REMARK (REporting recommendations for tumour MARKer prognostic studies) criteria for tumour marker studies [Sauerbrei et al., 2018],Sixty patients enrolled in the prospective study at the Department of Pulmonology, 1stDivision Internal Medicine, Clinical Centre, University of Pecs (Pecs, Hungary) between 01.02.2018 and 15.12.2018. Each participant signed an informed consent form and blood serum samples were collected before any treatment began. After the diagnosis of pulmonary carcinoma, rule-based therapy and data collection followed. Where surgery was possible, tissue samples were also collected and stored at the Department of Pathology, Clinical Centre, University of Pecs (Pecs, Hungary). The inclusion criteria were as follows: (i) histologically confirmed NSCLC stage l-IV (LUAD or LUSC) based on American Joint Committee on Cancer (AJCC, version 8) staging; (ii) no history of radiotherapy, immunotherapy, chemotherapy, or other treatments before diagnosis; (iii) availability of complete follow-up data including best overall response (BOR) and overall survival (OS); and (iv) availability of sufficient quantity and quality of serum samples. The exclusion criteria were as follows: (i) pathologically different diagnoses of NSCLC; (ii) patients with a history of a second primary malignancy; and (iii) serum samples or any other reason for the failure of quality control at any stage of the study. BOR was determined according to RECIST version 1.1 [Therasse et al., 2000], The time from the start of treatment to the end of any cause or the last follow-up date was defined as OS. Serum from healthy blood donors (HC) selected by a comparable age distribution to the study population were used as controls. The study was declared complete three years later, in 2021. The study was approved by the local Research Ethics Committee, University of Pecs (PTE_KK_RIKEB_6444 / 2016) and was conducted in compliance with the Declaration of Helsinki.

[0248] Ethics approval and consent to participate:

[0249] This study was carried out in accordance with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the University of Pecs (Date2016 / No6444).

[0250] Written informed consent was obtained from all individual participants in the study.

[0251] Out of the 60 patients who had verified primary lung carcinoma, 46 patients had complete follow-up data over the three-year study. Out of the 46 patients, only 24 (52.2%) patients had suitable quality and quantity of pretreatment serum samples. The detailed study flow according to REMARK criteria is described in Figure 1A and Table 4, see at the end of the examples, summarises patient baseline characteristics and the eventually applied therapy [Novello et al., 2016],

[0252] Out of the 24 NSCLC patients, the LUAD and LUSC subtypes represented 58.3% and 41.7%, respectively. Eight (33.3%) of the 24 patients were diagnosed with stage l-IIIA NSCLC, and 16 (66.67%) were at stage IIIB-IV. Additionally, most of the patients presented positive nodal status (78.3%) and had distant metastasis (58.3%). Based on the initial clinical parameters, Fisher's exact and chi-squared tests were performed, and the two subtypes were comparable for baseline characteristics (Supplementary Table 1). BOR was evaluated in 22 patients. From 14 LUAD patients, 4 (28.6%) showed an objective response [complete response (CR) + partial response (PR)]. SD was the best therapeutic outcome in 6 patients (42.9%), and 4 patients (28.6%) had PD. LUAD patients exited after a median survival time of 579 days. By contrast, of 10 LUSC patients, none of them had an objective therapy response, while 5 patients (50%) had SD, and 3 patients (30%) had PD. LUSC patients exited after a median survival time of 280 days.TOTAL LUAD LUSC p

[0253] n=14 n=10

[0254] Mean age 69 67

[0255]

[0256] Gender 0.4212A

[0257] Male 7 7

[0258] Female 7 3

[0259] Smoking history 0.3519B

[0260] Non-smoker 2 0

[0261] Former- smoker 7 5

[0262] Smoker 4 5

[0263] TNM stage 0.9999A

[0264] l-IIIA 5 3

[0265] IIIB-IV 9 7

[0266] Tumor invasion 0.4015A

[0267] T1-T27 3

[0268] T3-T46 7

[0269] Lymphatic invasion 0.3394A

[0270] No 4 1

[0271] N1-N39 9

[0272] Distant metastasis 0.9999A

[0273] M06 4

[0274] M18 6

[0275] Table 1. Patient characteristics at pre-treatment baseline. Statistical analyses were performed using Fisher's exact (A) and chi-square (B) tests.

[0276] EXAMPLE 3 - Used methods and equipment

[0277] Immunohistochemistry

[0278] Immunohistochemistry was performed on 4 μm thick tissue sections of FFPE tissue blocks. Tris / EDTA buffer, pH 9.0, was used for 20 min for antigen retrieval. Reactions were visualised via BOND polymer refine detection (Leica DS9800) (Leica Biosystems, Deer Park, IL, United States). A monoclonal antibody WNT5A (Clone 3D10) (MA5-15511, Thermo Fisher Scientific, Waltham, MA, United States) was used. Extraction of exosomes from serum

[0279] Blood samples were collected, allowed to clot at 37°C for 20 min, and then centrifuged at 1500×g for 10 min at room temperature (RT). The serum was stored at -80°C until further processing. Exosomes were isolated from 400 μl of serum samples using Total Exosome Isolation Reagent (TEI) (from serum) (4478360, Invitrogen, Thermo-Fisher Scientific, Waltham, MA, United States). Briefly, serum samples were spun at 2000× g for 30 min to remove cells and debris. Next, 0.2 volumes of TEI reagent were added to each supernatant, and the samples were incubated at 4°C for 30 min. The precipitated exosomes were recovered by centrifugation at 10,000×g for 10 min at RT. The exosome pellets were subsequently resuspended in PBS, pH 7.4, at RT.Nanoparticle tracking analysis

[0280] A NanoSight NS300 instrument (Malvern Panalytical Ltd., Malvern, United Kingdom) equipped with a 488-nm blue laser was used for real-time tracking and analysis.

[0281] All analysed samples were diluted in Ca- and Mg-free PBS to a final volume of 1 ml. Exosome isolates were subsequently diluted to the optimum NTA detection range before measurements (10-50 particles / frame). For each measurement, five 1-minute videos were captured under the following conditions: cell temperature: 25°C; syringe speed: 50 μl / s. The videos were analysed using the in-built NTA v3.2 software. Transmission electron microscopy (TEM)

[0282] TEM was used to visualise the exosomes. A 2.5 μl sample volume was placed individually on a 300-mesh grid of each sample. The grid was dried overnight at RT and then 5% uranyl acetate and 3% sodium citrate were added to the grid. After 5 min of incubation, the grid was air-dried. Twenty-four hours later, the grid was analysed using JEOLTEM (JEOL Ltd., Tokyo, Japan) 1,200 EX.

[0283] EV Antibody array

[0284] EV-specific marker analysis was performed using the Exo-Check antibody array (EXORAY210B-8, System Biosciences, Palo Alto, CA, USA). 60 pg of EV preparation was added to the membrane-based blot array, and the manufacturer's instructions were followed. The intensity of chemiluminescence was detected with a G: BOX Chemi XRQ. (Syngene, Cambridge, UK).

[0285] WNT5A ELISA

[0286] A human WNT5A (protein Wnt-5a) ELISA Kit (EH1164, Fine-Test, Wuhan, China) was used to quantify the serum, exosome-free serum, and exosome WNT5A contents. The starting volume was 400 μl of serum in all cases. To study the association and distribution of WNT5A with exosomes, the isolated pellet was resuspended in 495 μl of PBS to quantify the WNT5A content on the surface of exosomes. To determine the total WNT5A content of exosomes, the samples were resuspended in 400 μl PBS, then treated with 95 μl of ice-cold RIPA buffer (89900, Pierce RIPA buffer, Thermo-Fisher Scientific, Waltham, MA, United States) to disrupt the exosome membranes. All samples were incubated with 5 μl of 100x Halt-Protease inhibitor cocktail (87786, Thermo-Fisher Scientific, Waltham, MA, United States). The samples were mixed with their respective buffers and incubated on ice for 15 min. The protein concentration was quantified by a BCA assay using the Pierce™ BCA Protein Assay Kit (23225, Thermo-Fisher Scientific, Waltham, MA, United States) according to the manufacturer's instructions.

[0287] All reagents, standards, and samples were prepared according to the manufacturer's instructions. The absorbance of the samples was recorded at 450 nm via a Perkin Elmer Enspire Multiplate-Reader (Perkin Elmer, Waltham, MA, USA). The target concentrations were interpolated from a standard curve created from the standards with predefined concentrations. Each sample was measured in duplicate. The intraassay variation coefficient was 4.1%, whereas the inter-assay variation coefficient was 14%. Evaluation of the ELISA was conducted without the knowledge of the clinical data to ensure objectivity.

[0288] WNT5A protein per particle

[0289] The amount of WNT5A protein / particle was calculated as follows:

[0290] WNT5A concentration measured by ELISA (pg / mL)

[0291] The amount of WNT5A protein / particle= Particle concentration detected by NTA (particles / mL) (formula 1)Statistical analysis

[0292] Data are expressed as the means ± SDs. The normal distribution was tested by Shapiro-Wilk test. The Mann-Whitney U test, the Fisher's exact test, and the unpaired t-test were used for the comparison of two specifications. The Kruskal-Wallis test, one-way ANOVA and the chi-squared test was used for comparison of three or more specifications. Cut-off values for serum, exosome-free serum, and exosome WNT5A were determined by receiver operating characteristics (ROC) curve analysis or the best cut-off method, a web-based survival analysis tool tailored for medical research (KMplot) [Lánczky et al., 2021], Briefly, all possible cut-off values between the lower and upper quartiles are computed, and the bestperforming threshold is used as a cut-off. The Kaplan-Meier method was used to generate survival curves based on the length of time between primary treatment and exit. The log-rank test (Mantel-Cox) was used to compare the survival distributions. Multivariable analysis was carried out by the Cox proportional hazards model. For all analyses, a two-sided p<0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 9.0.0 software (GraphPad, Palo Alto, USA) or R (The R Foundation for Statistical Computing, Vienna, Austria).

[0293] EXAMPLE 4 - Exosome studies - quality assessment, exosome number as a diagnostic marker

[0294] To investigate the amount of WNT5A in vesicle-free and vesicle-bound fractions, WNT5A levels were measured in total serum, exosome-free serum, and serum-derived exosomes. The studied patient cohort was not in therapy at the time of serum sampling (Figure 1A-B). Healthy volunteers provided the control serum samples (HC). The quality of exosomes was determined by the morphology and size of exosomes, and the detection of exosome-specific markers (Figure 2A-F). NTA revealed not only that the exosome quality was suitable for further investigation but also that the circulating exosome number was greater in LUAD patients than in either HCs (1.6-fold) or in LUSC patients (2.2-fold). Whereas the exosome concentration in LUSC patients was not just lower than in LUAD patients but even lower than in HCs (0.72-fold).

[0295] WNT5A in blood serum and serum exosomes

[0296] Figure 3 displays the amount of WNT5A in total serum, exosome-free fraction of the serum, and exosomes both on the exosome surface and in the exosome lumen as cargo. Statistical significance was not detected between total serum WNT5A and exosome-depleted serum WNT5A (Figure 3A). However, it revealed that significantly less WNT5A protein is transported in exosomes than in the exosome-free fraction (p<0.0001)(Figure 3A). To further examine the distribution and proportion of WNT5A in exosomes, the isolated exosomes were resuspended in RIPA buffer to disrupt the exosome membrane and to measure WNT5A levels as a combination of surface and lumen (cargo) WNT5A (Figure 3A). When the exosome lumen and the exosome surface were measured together, a higher level of WNT5A protein was detected, although the increase was not statistically significant (Figure 3A).

[0297] Elevated WNT5A levels were detected in NSCLC patients compared to HCs in all examined serum fractions (Figure 3B). However, when the total serum and exosome-free serum of LUAD and LUSC patients were analysed separately, a significant difference between the two subtypes was detected only in the exosome-free serum fraction (Figure 3C, 3D), where LUSC samples contained less soluble WNT5A compared to LUAD (p=0.051) (Figure 3D).Considering that LUAD patients' sera had significantly higher particle numbers than LUSC patients (Figure 2), pg / particle WNT5A levels were calculated as well to measure the level of WNT5A / exosomes. The results revealed that the exosomes isolated from LUSC samples carry significantly greater amounts of WNT5A in their lumen and on their surface (Figure 3E) than either HCs or LUAD exosomes do. Similarly to exosome-free serum fractions, we found a significant difference between the total WNT5A exosome content of patients with LUAD and LUSC (exosome surface+cargo, Figure 3F). WNT5A content / particle in LUAD and HC exosomes did not differ significantly (Figure 3E-F).

[0298] WNT5A as a biological marker

[0299] To ensure the results are applicable for clinical use, we determined the optimal cut-off values of serum, exosome-free serum, normalized surface exosome, and normalized surface+cargo exosome WNT5A levels. The cut-off value of WNT5A in the exosome-free serum was 740 pg / mL. Cut-off values were also calculated for WNT5A / particle number. On exosome surface, 1.8×10-9pg of WNT5A / NP was determined as cut-off value, while 2.5×10-9pg WNT5A / NP for exosome surface+cargo.

[0300] In patients with low levels of WNT5A in exosome-free serum (<740 pg / mL), the OS was shorter compared to those with higher WNT5A levels (>740 pg / mL) (logrank p=0.0042; HR=0.23; 95% Cl 0.08 -0.68; Figure 4A). Conversely, patients with low exosome surface+cargo WNT5A levels (< 2.5×10-9pg WNT5A / NP) had a longer O / S compared to those with high WNT5A levels (>2.5×10-9pg WNT5A / NP) in their exosomes (logrank p=0.028, HR= 2.95; 95% Cl 1.08-8.08; Figure 4B). In addition, univariable analysis revealed that histology (p=0.03, HR= 3.1; 95% Cl 1.1-8.4) and TNM stage (p=0.02, HR=3.6; 95% Cl 1.3-10) were also a predictor of O / S. However, by multivariable analysis, only exosome-free serum (p=0.05, HR=4.58; 95% Cl 0.95-22.01) and TNM stage (p=0.01, HR=5.79; 95% Cl 1.63-20.62) were found as an independent predictor for O / S. (Figure 4C / D, Table 2).

[0301] Table 2 - Univariate Cox regression analysis and Multivariate Cox regression analysis of data

[0302] Univariate Cox regression analysis

[0303] Overall survival Parameters included Hazard ratio (95% Cl) P * Age (<60 versus >60 years) 0.77 (0.25-2.4) 0.65 Gender 2.4 (0.96-5.8) 0.06 Smoking history (Non or former-smoker versus smoker) 1(0.44-2.5) 0.92 Histology (LUAD versus LUSC) 3.1(1.1-8.4) 0.03 TNM stage (I-111A versus 111B-1V) 3.6 (1.3-10) 0.02 Exosome-free serum (<740 pg / mL versus= 740 pg / mL) 4.3 (1.5-13) 0.01 Exosome surface+ cargo (>2.5x10° pg / NP versus = >2.5x10° pg / NP) 0.34(0.12-0.93) 0.04 Multivariate Cox regression analysis

[0304] Overall survival Parameters included Hazard ratio (95% Cll) P Age (=60 versus >60 years) 1.07 (0.28-4.08) 0.92 Gender 2.13 (0.61-7.4) 0.24 Smoking history N( on or former-smoker versus Smoker) 0.82(0.30-2.25) 0.70 TNM stage (I-111A versus III 1B-1V) 5.79(1.63-20.62) 0.01 Exosome-free serum ( < 740 pg / mL versus = 740 pg / mL) 4.58 (0.95-22.01) 0.05 Exosome surface+ cargo (> 2.5x10° pg / NP versus = >2.5x10° pg / NP) 1.47(0.35-6.22) 0.60

[0305]

[0306] Table 3 - Cut-off values of WNT5A are indicated by ROC curve analysis in each group.

[0307] WNT5A

[0308] Serum Exosome-free serum

[0309] NSCLC HC NSCLC HC LUAD LUSC

[0310] Boundary line

[0311] >656 <656 >515 <515 > 737 515 >x<737 (pg / mL)

[0312] 0.869 0.9702 0.7714

[0313] AUC (95% Cl)

[0314] (0.6728 to 1.000) (0.9099 to 1.000) (0.5667 to 0.9762) Sensitivity % 91.67 95.83 90 Specificity % 85.71 100 71.43

[0315] P-value 0.0034 0.0002 0.0261

[0316] Exosome surface Exosome surface+cargo

[0317] NSCLC HC NSCLC HC LUAD LUSC

[0318] Boundary line

[0319] >9.1x10-10<9.1x10-10>1.35xl0’9<1.35xl0’9<2.45x10-9>2.45x10-9(pg / NP)

[0320] 0.7857 0.9018 0.9286

[0321] AUC (95% Cl)

[0322] (0.5760 to 0.9954) (0.7708 to 1.000) (0.8194 to 1.000) Sensitivity % 71.43 83.33 90 Specificity % 85.71 85.71 92.86

[0323] P-value 0.00367 0.0014 0.0004

[0324]

[0325] Furthermore, elevated WNT5A / particle on the exosome surface at the therapy baseline was associated with distant metastasis in patients with LUSC (p = 0.0056, Figure 4E). Consequently, more advanced stages of the disease (p=0.0118) (Figure 4F) and lymph node involvement (p=0.0443) (Figure 4G) were significantly associated with elevated levels of WNT5A on particle surfaces (>1.8×10-9pg of WNT5A / NP). Additionally, a high level of WNT5Aon the exosome surface / particle (>1.8×10-9pg of WNT5A / NP) can also predict a lack of response to therapy in LUSC patients (p=0.0073) (Figure 4H).

[0326] In contrast, no such correlations were detected in LUAD patients in any of the examined parameters, indicating that elevated WNT5A on the surface of exosomes only plays a significant role in the LUSC subtype. Supplementary Table 2 demonstrates the ROC curve evaluation of the diagnostic efficacy of different fractions of extracellular WNT5A. The exosome-free serum WNT5A yielded the highest AUC value:0.9702, [Cl]: 0.9099 to 1.000) comparing NSCLC patients with the HC group, separating cancer patients from healthy donors. The second highest AUC value was calculated for WNT5A in exosome surface+cargo, which the most accurately distinguished LUAD and LUSC subtypes (AUC: 0.9286, [Cl]: 0.8194 to 1.000, Table 3).

[0327] Table 4 below summarizes the patient baseline characteristics and the eventually applied therapy.Cftaractonstics M

[0328] Age (years)

[0329] ” < so ’ 4 nw

[0330] > 60 20 (83.33)

[0331]

[0332] Gsftder

[0333] Mate 14(58.33)

[0334] Fsteteto 18(41.87)

[0335] Smoking history

[0336] htoft-'S rritOt 2 (8. ) j

[0337] FsxfiW smote 12 (52.17)

[0338] Smoker § (39.13)

[0339] Histology

[0340] Squamous catotoma 0. USC) 10 (41.67)

[0341] AOehOtWClm (LOAD) 14 (§8.33)

[0342] W! stage

[0343] MliA S (33.33;

[0344] UiB-IV 18(88.67)

[0345] llsmour Invasion

[0346] T1-T2 10(43.48)

[0347] T3-T4 13(58.52)

[0348] Lymphatic invasion

[0349] HO 5 (21.74)

[0350] H 3 18(78.26)

[0351] Distant metastasis

[0352] M0 18(41.67)

[0353] Ml 14 (88.33)

[0354] Treatments of LUSC

[0355] CDDP-GEM & (90.3)

[0356]

[0357] OSP-GEM f(io.o)

[0358] Trsatmsfits of LOAD

[0359] CDDP-FSM 6 (42.88)

[0360] CDDP-TXT 2 (14,23)

[0361] CDSP-Pacteaxel 1 (7.14)

[0362] CBF-PEM 1 (7.14)

[0363] CBP *acgtawi 3 (21.43)

[0364] CHP'PaciitaxeHAvsstin 1 (7.14)

[0365] Dissas® Ssags aeec Sfog to to>s 8* edito Lung Ga er TN Staging,

[0366] TH < tosaosf / i-sodatoisstastosis; DDD Oispteito; GEM, G®s»x;itabte®; COP,

[0367] Carttopiatta: Pf? M, Pe eirexed:: TXT, LkteStaxei

[0368] Table 4 - Summary of patient baseline characteristics and the eventually applied therapy ADVANTAGES AND INDUSTRIAL APPLICABILITY

[0369] The invention can serve as the basis of further studies, that are needed to elucidate the underlying selection and sorting process of WNT5A into extracellular space and vesicles to elucidate the distinctively different effects on survival. Understanding such differences might also lead to the identification of novel therapeutic targets, which are especially important since exosome-associated high WNT5A level is an indicator of aggressive disease with higher metastatic potential and reduced O / S [Zhang et al., 2017], It is also significant, both clinically and financially, that from a simple blood sample and by measuring WNT5A in serum and exosome fractions at the time of patient admission we can identify advanced disease stage, lymph node involvement, distant metastasis, and predict lack of therapy response, and reduced expectable survival time associated with LUSC.

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[0416]

Claims

1. CLAIMS1. A method for differentiating between subtypes in non-small cell lung cancer (NSCLC) patients, the method comprising:- measuring the exosome concentration in a serum sample comprising exosomes obtained from / of an NSCLC patient, and- measuring the normalized exosome-bound WNT5A level in serum-derived exosomes, preferably the WNT5A content of the exosomes relative to the exosome concentration in a serum sample comprising exosomes obtained from / of an NSCLC patient, and- determining the NSCLC subtype for the patient based on the measured exosome-bound WNT5A level of circulating exosomes, wherein if the exosome-bound WNT5A level is higher than a pre-determined threshold value, the NSCLC is considered as lung squamous cell carcinoma (LUSC), and, if the exosome-bound WNT5A level is lower than a pre-determined threshold value, the NSCLC is considered as lung adenocarcinoma (LUAD).

2. The method of claim 1 whereinthe normalized exosome-bound WNT5A level is the WNT5A content of the exosomes relative to the exosome concentration in the serum, optionally expressed as WNT5A content per exosomes, and the pre-determined threshold level is cut-off value for the exosome-bound WNT5A level differentiating between LUSC and LUAD,wherein in the determining stepthe NSCLC is considered as LUSC if the elevated exosome-bound WNT5A level is higher than the cut-off value for the exosome-bound WNT5A level differentiating between LUSC and LUAD, and preferably, the NSCLC is considered as LUAD if the elevated exosome-bound WNT5A level is not higher (preferably lower) than a cut-off value for the exosome-bound WNT5A level differentiating between LUSC and LUAD.

3. The method of claim 2, wherein in the determining step the cut-off value for the exosome-bound WNT5A level differentiating between LUSC and LUAD is expressed in WNT5A content per exosome nanoparticle, and is between 2 x IO-9and 3 x IO-9WNT5A per exosome nanoparticle, preferably between 2,2-2,8 x 10-9WNT5A per exosome nanoparticle, more preferably between 2,4-2, 6 x 10'9WNT5A per exosome nanoparticle.

4. The method of any of claims 1 to 3 wherein the exosome-bound WNT5A level in circulating exosomes comprises or is the WNT5A level on the surface of the exosomes.

5. The method of any of claims 1 to 3, wherein the exosome-bound WNT5A level in circulating exosomes comprises boththe WNT5A level on the surface of the exosomes andthe WNT5A level in the lumen of the exosomes (cargo).

6. The method of any of claims 4 to 5 wherein the WNT5A level on the surface of the exosomes is measured by an antibody binding based method to measure the WNT5A level and a method to assess the number of exosomes.

7. The method of any of claims 4 to 5 whereinthe WNT5A level in the lumen of the exosomes is measured by a method comprising an antibody binding based method to measure the WNT5A level and a method to assess the number of exosomes, preferably ELISA and NTA (Nanoparticle tracking analysis)and optionally TEM (Transmission electron microscopy plus EV Antibody array.

8. The method of any of claims 1 to 7 wherein the method also comprises- predicting disease progression of an NSCLC patient based on the exosome-bound WNT5A level, wherein an elevated exosome-bound WNT5A level indicates a shorter expectable survival time and a decreased exosome-bound WNT5A level indicates longer expectable survival time.

9. The method of claim 8, further comprising- determining disease progression,preferably metastasis level and lymph node involvement of an NSCLC patient having the subtype LUSC, wherein the elevated exosome-bound WNT5A level is the level of exosome-surface bound WNT5A protein level measured on the circulating exosomes obtained from a serum sample of the patient, wherein the elevated exosome-surface bound WNT5A protein level compared to the normal, preferably median level among NSCLC patients having the subtype LUSC indicates a progressed disease with a high distant metastasis chance and lymph node involvement.wherein optionally a therapy is proposed depending on the determined disease progression of the patient.

10. The method of claim 9, whereinthe measurement is carried out at therapy baseline, and- elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) at the therapy baseline is indicative of distant metastasis in patients with LUSC and / or- elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is indicative of advanced stage of the disease, and / or- elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is indicative of lymph node involvement, and / or- elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is predictive of a lack of response to therapy in LUSC patients.wherein optionally a therapy is proposed depending on the determined disease progression of the patient.

11. A method for predicting disease progression in non-small cell lung cancer (NSCLC) patients, the method comprising:- measuring the exosome-bound WNT5A level in circulating exosomes, obtained from a serum sample of an NSCLC patient preferably the WNT5A content of the exosomes relative to the exosome concentration in a sample comprising exosomes obtained from / of an NSCLC patient);- predicting disease progression, preferably by the expectable survival time of an NSCLC patient based on the exosome-bound WNT5A level,wherein an elevated exosome-bound WNT5A level indicates a short expectable survival time and a decreased exosome-bound WNT5A level indicates long expectable survival time.wherein if the exosome-bound WNT5A level is higher than a pre-determined threshold value, it indicates a short expectable survival time and, if the exosome-bound WNT5A level is lower than a pre-determined threshold value, it indicates long expectable survival time.wherein optionally a therapy is proposed depending on the predicted disease progression of the patient.

12. The method of claim 11, whereinthe normalized exosome-bound WNT5A level is the WNT5A content of the exosomes relative to the exosome concentration in the serum, optionally expressed as WNT5A content per exosomes, and the pre-determined threshold level is a cut-off value for differentiating between a short expectable survival time and a long expectable survival time, preferably the same as the exosome-bound WNT5A level differentiating between LUSC and LUAD,wherein in the determining stepthe NSCLC patient is considered as having a short expectable survival time if the elevated exosome-bound WNT5A level is higher than the cut-off value for differentiating between a short expectable survival time and a long expectable survival time, and preferably,the NSCLC patient is considered as having a short expectable survival time, if the elevated exosome-bound WNT5A level is not higher (preferably lower) than a cut-off value for the exosome-bound WNT5A level differentiating between LUSC and LUAD.

13. The method of claim 12, whereinthe exosome-bound WNT5A level is expressed as WNT5A content per exosome nanoparticle, and in the determining stepthe cut-off value for WNT5A content per exosomes differentiating between a short expectable survival time and a long expectable survival time is between 2-3 x 10-9picograms of WNT5A per exosome (nanoparticle), preferably between 2,2-2,8 x 10-9picograms of WNT5A per exosome (nanoparticle), more preferably between 2,4-2,6 x 10-9picograms of WNT5A per exosome (nanoparticle).

14. The method of any of claims 11 to 13 whereinthe exosome-bound WNT5A level in circulating exosomes comprises or is the WNT5A level on the surface of the exosomes.

15. The method of claim 15 wherein the exosome-bound WNT5A level in circulating exosomes comprises both the WNT5A level on the surface of the exosomes and the WNT5A level in the lumen of the exosomes.

16. The method of any of claims 11 to 15, further comprising- determining disease progression,preferably metastasis level and lymph node involvement, of an NSCLC patient having short expectable survival time and preferably having the subtype LUSC, based on the level of exosome-surface bound WNT5A protein measured on the circulating exosomes obtained from a serum sample of the patient, wherein an elevated exosome-surface bound WNT5A protein level compared to the median level among NSCLC patients having the subtype LUSC indicates a progressed disease with a high chance of distant metastasis and lymph node involvement,and optionally proposing a therapy corresponding to disease progression of the patient.

17. The method of any of claims 11 to 16 further comprising predicting treatment response of an NSCLC patient having the subtype LUSC, based on the level of exosome-surface bound WNT5A protein measured on the circulating exosomes obtained from a serum sample of the patient, wherein a decreased level of exosome-surface bound WNT5A predicts a good response to treatment and an elevated level of exosome-surface bound WNT5A protein predicts a bad response to treatment, andoptionally proposing a therapy corresponding to the predicted treatment response of the patient.

18. The method of any of claims 11 to 17, whereinthe measurement is carried out at therapy baseline, and- elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) at the therapy baseline is indicative of distant metastasis in patients with LUSC and / or- elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is indicative of advanced stage of the disease, and / or- elevated WNT5A / particle on the exosome surface is (elevated level of WNT5A on particle surfaces) indicative of lymph node involvement, and / or- elevated WNT5A / particle on the exosome surface (elevated level of WNT5A on particle surfaces) is predictive of a lack of response to therapy in LUSC patients,wherein optionally a therapy is proposed depending on the determined disease progression of the patient.

19. The method of any of claims 11 to 18, further comprising- differentiating between progressive disease and stable disease of an NSCLC patient having the subtype LUSC, based on the level of exosome-surface bound WNT5A protein measured on the circulating exosomes obtained from a serum sample of the patient, wherein a decreased level of exosome-surface bound WNT5A predicts a stable disease (LUSC SD) and an elevated level of exosome-surface bound WNT5A protein predicts a progressive disease (LUSC PD), andand optionally proposing a therapy corresponding to the predicted treatment response of the patient.

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