EP4642933A1

EP4642933A1 - Compositions, kits and methods for determining personalized treatment regimen

Info

Publication number: EP4642933A1
Application number: EP23911158.6A
Authority: EP
Inventors: Limor BRODAY; Amir ONN
Original assignee: Ramot at Tel Aviv University Ltd; Sheba Impact Ltd
Current assignee: Ramot at Tel Aviv University Ltd; Sheba Impact Ltd
Priority date: 2022-12-28
Filing date: 2023-12-28
Publication date: 2025-11-05
Also published as: WO2024142066A1

Abstract

The present disclosure provides composition and kit for stabilization and characterization of cfDNA in biological samples and methods for determining personalized medicine.

Description

COMPOSITIONS, KITS AND METHODS FOR DETERMINING PERSONALIZED TREATMENT REGIMEN

TECHNOLOGICAL FIELD

The invention provides compositions, kits and methods for determining personalized treatment in a subject suffering from a pathologic disorder.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

[1] Husain H, Melnikova VO, Kosco K, Woodward B, More S, Pingle SC, Weihe E, Park BH, Tewari M, Erlander MG, Cohen E, Lippman SM, Kurzrock R. Monitoring Daily Dynamics of Early Tumor Response to Targeted Therapy by Detecting Circulating Tumor DNA in Urine. Clin Cancer Res. 2017 Aug 15;23(16):4716-4723.

[2] Catarino R, Coelho A, Araujo A, Gomes M, Nogueira A, Lopes C, Medeiros R. Circulating DNA: diagnostic tool and predictive marker for overall survival of NSCLC patients. PLoS One. 2012;7(6):e38559. doi: 10.1371/journal.pone.0038559.

[3] Nygaard AD , Holdgaard PC , Spindler KLG , Pallisgaard N , Jakobsen A . The correlation between cell-free DNA and tumour burden was estimated by PET/CT in patients with advanced NSCLC. Br J Cancer 2014;110:363-8.

[4] Tissot C , Toffart AC , Villar S , Souquet PJ , Merle P , Moro-Sibilot D , et al . Circulating free DNA concentration is an independent prognostic biomarker in lung cancer. Eur Respir J 2015;46: 1773-80.

[5] Hyun MH , Sung JS , Kang EJ , Choi YJ , Park KH , Shin SW , et al . Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer. Oncotarget 2017;8:94417-30.

[6] Rafal Dziadziuszko, Solange Peters, Tony Mok, D Ross Camidge, Shirish M Gadgeel, Sai-Hong Ignatius Ou, Krzysztof Konopa, Johannes Noe , Malgorzata Nowicka, Walter Bordogna, Peter N Morcos, Vlatka Smoljanovic, Alice T Shaw Circulating Cell- free DNA as a Prognostic Biomarker in Patients with Advanced ALK+ Non-small Cell Lung Cancer in the Global Phase III ALEX Trial, Clinical Cancer Research 2022 DOI: 10.1158/1078-0432.CCR-21-2840

[7] Moiseyenko, F.V., Kuligina, E.S., Zhabina, A.S. et al. Changes in the concentration of EGFR-mutated plasma DNA in the first hours of targeted therapy allow the prediction of tumor response in patients with EGFR-driven lung cancer. Int J Clin Oncol 27, 850-862 (2022). https://doi.org/10.1007/sl0147-022-02128-6

[8] Riediger AE, Dietz S, Schirmer U, Meister M, Heinzmann-Groth I, Schneider M, Muley T, Thomas M, Siiltmann H. Mutation analysis of circulating plasma DNA to determine response to EGFR tyrosine kinase inhibitor therapy of lung adenocarcinoma patients. Sci Rep. 2016 Sep 19; 6:33505. doi: 10.1038/srep33505. PMID: 27640882; PMCID: PMC5027592.

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

Monitoring disease progression and response to treatment is mostly done by imaging techniques including computed tomography (CT), positron emission tomography (PET)-CT and magnetic resonance imaging (MRI) scans. Yet, the use of such technique is limited as they are done several weeks after treatment initiation and in addition lack information regarding molecular and pathologic changes occurring during therapy.

Modulation of cell-free DNA (cfDNA) levels were previously shown in various pathological conditions [1]. For example, an increase in baseline cfDNA concentration has been shown to be associated with poor prognosis in non-small cell lung cancer (NSCLC) [2-5]. In addition, a recent retrospective analysis of data from the ALEX clinical study used plasma cfDNA concentration to assess prognosis of advanced anaplastic lymphoma kinase (ALK)⁺ NSCLC [6]. Further, several studies in NSCLC patients showed that transient elevation in circulating tumor DNA (ctDNA) levels immediately following treatment initiation correlated with tumor response to therapy [7,8].

GENERAL DESCRIPTION

In accordance with some aspects, the present disclosure provides a composition comprising means to bind cell free DNA (cfDNA) and/or one or more cfDNA stabilizing reagents.

In some embodiments, the one or more cfDNA stabilizing reagents is or comprise at least one DNase inhibitor. In some embodiments, the at least one DNase inhibitor is actin. In some embodiments, the one or more cfDNA stabilizing reagents is or comprise actin and an actin filament inhibitor. In some embodiments, the one or more cfDNA stabilizing reagents is or comprise actin and latrunculin. In some embodiments, the composition comprises means to bind cfDNA. In some embodiments, the means to bind cfDNA is or comprises magnetic beads.

In accordance with some aspects, the present disclosure provides a composition comprising means to bind cfDNA and/or one or more cfDNA stabilizing reagents for use in stabilizing cfDNA in a biological sample or fraction thereof. In some embodiments, stabilizing cfDNA or a faction thereof from a biological sample is for at most 10 days.

In accordance with some aspects, the present disclosure provides a kit comprising a one or more container means, at least one of the one or more container means comprises a composition comprising one or more means to bind cfDNA and/or one or more cfDNA stabilization reagents, and optionally comprising instructions for preparing one or more urine samples for cfDNA characterization.

The present disclosure provides in accordance with some aspects, a method comprising contacting one or more urine samples with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, the one or more urine samples have been collected from a subject.

The present disclosure provides in accordance with some aspects, a method comprising collecting one or more urine samples from a subject and contacting each one of the a one or more urine samples with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents.

The present disclosure provides in accordance with some aspects, a method of preparing at least one urine sample for characterizing cfDNA in the sample, the method comprising collecting the at least one urine sample from a subject who received at least one dose of a treatment and contacting the at least one urine sample with cfDNA binding means and/or one or more cfDNA stabilizing reagents.

The present disclosure provides in accordance with some other aspects, a method of preparing urine samples for characterizing cfDNA in the urine samples, the method comprising (i) contacting at least one urine sample obtained from a subject at a first time point with cfDNA binding means and/or one or more cfDNA stabilizing reagents, (ii) repeating step (i) at a second time point, wherein the subject received at least one dose of a treatment between the first time point and the second time point.

The present disclosure provides in accordance with some further aspects, a method of determining cfDNA level in at least one urine sample, comprising contacting the at least one urine sample, any fraction thereof or any cfDNA obtained therefrom with at least one cfDNA specific detecting molecule, wherein the at least one urine sample is obtained from a subject who received at least one dose of a treatment.

The present disclosure provides in accordance with yet some other aspects, a method of determining cfDNA level in a urine sample, comprising (i) contacting at least one urine sample, any fraction thereof or any cfDNA obtained from a subject at a first time point with at least one cfDNA specific detecting molecule, (ii) repeating step (i) at a second time point, wherein the subject received at least one dose of a treatment between the first time point and the second time point.

The present disclosure provides in accordance with yet some further aspects, a method of determining cfDNA level in a urine sample, the method comprising: (i) contacting at least one urine sample with cfDNA binding means and/or one or more cfDNA stabilizing reagents and (ii) contacting the at least one urine sample, any fraction thereof or any cfDNA obtained therefrom with at least one cfDNA specific detecting molecule, wherein the at least one urine sample is obtained from a subject who received at least one dose of a treatment.

The present disclosure provides in accordance with some other aspects, a method of determining cfDNA level in a urine sample, the method comprising: (i) contacting at least one urine sample with cfDNA binding means and/or one or more cfDNA stabilizing reagents and contacting the at least one urine sample, any fraction thereof or any cfDNA obtained therefrom with at least one cfDNA specific detecting molecule, (ii) repeating step (i) at a second time point, wherein the subject received at least one dose of a treatment between the first time point and the second time point.

The present disclosure provides in accordance with yet some other aspects, a method for determining treatment efficacy in a subject, comprising: determining level of cfDNA in at least two urine samples, wherein at least one urine sample of the at least two urine samples is obtained from the subject at a first time point and at least one urine sample of the at least two urine samples is obtained from the subject at a second time point, wherein the subject received at least one dose of the treatment between the first time point and the second time point; and identifying the treatment as being effective in the subject if the change in cfDNA level determined in the second time point compared to the level determined in the first time point is above a predetermined change.

The present disclosure provides in accordance with some additional aspects, a method for assessing responsiveness of a subject to a treatment, the method comprising: (a) determining level of cfDNA in at least two temporally- separated urine samples obtained from the subject, wherein at least one urine sample is obtained from the subject before the subject received at least one dose of the treatment and the temporally- separated urine sample is obtained from the subject after said subject received at least one dose of the treatment, and (b) calculating a rate of change of the level of cfDNA between said samples; wherein responsiveness to the treatment is associated with a rate of change above a predetermined rate of change.

The present disclosure provides in accordance with yet some additional aspects, a method for assessing responsiveness of a subject to a treatment regimen, the comprises the steps of: (i) determining level of cfDNA in at least one urine sample obtained from the subject at a time point before the subject received at least one dose of the treatment,

(ii) determining level of cfDNA in at least one urine sample obtained from the subject at a time point after the subject received at least one dose of the treatment, and (iii) determining whether the subject is responsiveness to treatment regimen.

The present disclosure further provided in accordance with some aspects, a method for assessing responsiveness of a subject to a treatment regimen, the method comprises the steps of: (i) determining level of cfDNA in at least one urine sample obtained from the subject at a time point before the subject received at least one dose of the treatment, (ii) determining level of cfDNA in at least one urine sample obtained from the subject at a time point after the subject received at least one dose of the treatment, and

(iii) calculating the rate of change of the level of cfDNA between the at least one sample obtained at a time point prior to the treatment and the at least one sample obtained at a time point after initiation of the treatment; wherein a rate of change below a predetermined rate of change indicates that the subject belongs to a pre-established non- responsive population.

Embodiments

Some embodiments of this disclosure will now be described in the following numbered paragraph. The following description intends to add on the above general description and not limit it in any manner.

1. A composition, a kit comprising one or more means to bind cfDNA and at least one cfDNA stabilization reagent.

2. The composition, the kit of Embodiment 1 for use in stabilizing total cfDNA in a biological sample.

3. The composition, the kit of Embodiment 1 or 2, wherein said biological sample is a urine sample.

4. The composition, the kit of Embodiment 2 or 3, wherein said cfDNA is stable for at most 2 weeks in the composition. 5. A method comprising contacting one or more urine samples with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, the one or more urine samples have been collected from a subject.

6. A method comprising collecting one or more urine samples from a subject and contacting each one of the a one or more urine samples with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents.

7. A method comprising filtering at least one urine sample to obtain a filtered urine sample and contacting the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, the one or more urine samples have been collected from a subject.

8. A method of preparing at least one urine sample, the method comprises the steps of collecting one or more urine samples from a subject, filtering the one or more urine samples to obtain a filtered urine sample and contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents.

9. A method of isolating cfDNA from at least one urine sample, the method comprises contacting the at least one urine sample collected from a subject with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, and isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA.

10. A method of isolating cfDNA from at least one urine sample, the method comprises collecting one or more urine samples from a subject, contacting each one of the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents and isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA.

11. A method of isolating cfDNA from at least one urine sample, the method comprises the steps of filtering at least one urine sample collected from a subject to obtain a filtered urine sample, contacting the at least one filtered urine sample with the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, and isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA.

12. A method of isolating cfDNA from at least one urine sample, the method comprises the steps of collecting one or more urine samples from a subject, filtering the one or more urine samples to obtained a filtered urine sample, contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents and isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA.

13. A method of characterizing cfDNA in at least one urine sample, the method comprises contacting the at least one urine sample collected from a subject with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA.

14. A method of characterizing cfDNA in at least one urine sample, the method comprises collecting one or more urine samples from a subject, contacting each one of the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA.

15. A method of characterizing cfDNA in at least one urine sample, the method comprises the steps of filtering at least one urine sample collected from a subject to obtain a filtered urine sample, contacting the at least one filtered urine sample with the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA.

16. A method of characterizing cfDNA in at least one urine sample, the method comprises the steps of collecting one or more urine samples from a subject, filtering the one or more urine samples to obtained a filtered urine sample, contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA.

17. The method of any one of the preceding Embodiments, comprising collecting at least one urine sample at a first time point and at least one urine sample at a second time point, wherein the subject received at least one dose of a treatment between the first time point and the second time point.

18. A method comprises contacting at least one urine sample collected from a subject at a first time point and at a second time point with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA and wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

19. A method comprises collecting one or more urine samples from a subject at a first time point and at a second time point, contacting each one of the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA and wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

20. A method comprises the steps of filtering at least one urine sample collected from a subject at a first time point and at a second time point to obtain a filtered urine sample, contacting the at least one filtered urine sample with the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA and wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

21. A method comprises the steps of collecting one or more urine samples from a subject at a first time point and at a second time point, filtering the one or more urine samples to obtained a filtered urine sample, contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA and wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

22. A method comprising contacting at least one urine sample collected from a subject at a first time point and at a second time point with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA, characterizing cfDNA and classifying the subject, wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

23. A method comprising collecting one or more urine samples from a subject at a first time point and at a second time point, contacting each one of the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA, characterizing cfDNA and classifying the subject, wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

24. A method comprising filtering at least one urine sample collected from a subject at a first time point and at a second time point to obtain a filtered urine sample, contacting the at least one filtered urine sample with the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA, characterizing cfDNA and classifying the subject, wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment. 25. A method comprising collecting one or more urine samples from a subject at a first time point and at a second time point, filtering the one or more urine samples to obtained a filtered urine sample, contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA, characterizing cfDNA and classifying the subject, wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

26. The method of any one of the preceding Embodiments, comprises classifying the subject as being responder to treatment if the cfDNA level determined at the second time point is higher (increased) as compared to the level determined at the first time point, wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

27. The method of any one of the preceding Embodiments, characterizing level of cfDNA in at least two urine samples and classifying the subject.

28. The composition, the kit, the method of any one of the preceding Embodiments, wherein said cfDNA is stable for at most 30 days, optionally at most 14 days, optionally at most 10 days, optionally at most 7 days in the composition.

29. The composition, the kit, the method of any one of the preceding Embodiments, wherein said cfDNA is stable for at most 7 days in the composition.

30. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more means to bind cfDNA is or comprises a solid support.

31. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more means to bind cfDNA is or comprises magnetic beads, super magnetic beads, magnetic nanoparticles or a combination thereof.

32. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more means to bind cfDNA is or comprises magnetic beads. 33. The composition, the kit, the method of any one of the preceding Embodiments, wherein said at least one cfDNA stabilization reagent is or comprises at least one DNase inhibitor.

34. The composition, the kit, the method of Embodiment 33, wherein the at least one DNase inhibitor is or comprises actin filament.

35. The composition, the kit, the method of any one of the preceding Embodiments, wherein said at least one cfDNA stabilization reagent is or comprises an actin filament inhibitor.

36. The composition, the kit, the method of Embodiment 35, wherein said actin filament inhibitor is at least one of substoichiometric cytochalasin B (CB), cytochalasin D, latrunculin or a combination thereof.

37. The composition, the kit, the method of Embodiment 34 or 35, wherein said actin filament inhibitor is latrunculin.

38. The composition, the kit, the method of any one of the preceding Embodiments, wherein said at least one cfDNA stabilization reagent is or comprises latrunculin.

39. The composition, the kit, the method of any one of the preceding Embodiments, comprising a salt.

40. The composition, the kit, the method of any one of the preceding Embodiments, comprising CaCh.

41. The composition, the kit, the method of any one of the preceding Embodiments, comprising at least one nucleotide.

42. The composition, the kit, the method of any one of the preceding Embodiments, comprising ATP.

43. The composition, the kit, the method of any one of the preceding Embodiments, comprising a sulfur containing compound.

44. The composition, the kit or the method of any one of the preceding Embodiments, comprising a reducing agent. 45. The composition, the kit, the method of any one of the preceding Embodiments, comprising a dithiol containing compound, a diol containing compound or a combination thereof.

46. The composition, the kit, the method of any one of the preceding Embodiments, comprising DTT.

47. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more means to bind cfDNA is or comprises magnetic beads in an amount of between about 2 pl and about 30 pl.

48. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more means to bind cfDNA is or comprises magnetic beads in an amount of between about 5 pl magnetic beads and about 25 pl magnetic beads.

49. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more means to bind cfDNA is or comprises magnetic beads in an amount of between about 10 pl magnetic beads and about 20 pl magnetic beads.

50. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more cfDNA stabilization reagents is or comprises actin filament in an amount of 50 ng/ml and about 20 pg/ml.

51. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more cfDNA stabilization reagents is or comprises actin filament in an amount of between about 1 pg/ml and about 8 pg/ml.

52. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more cfDNA stabilization reagents is or comprises actin filament in an amount of between about 0.5 pg/ml and about 8 pg/ml.

53. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more cfDNA stabilization reagents is or comprises latrunculin in an amount of between about IpM and about 150pM.

54. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more cfDNA stabilization reagents is or comprises latrunculin in an amount of between about 20 pM and about 150 pM. 55. The composition, the kit, the method of any one of the preceding Embodiments, wherein said one or more cfDNA stabilization reagents is or comprises latrunculin in an amount of between about 50 pM and about 150 pM.

56. The composition, the kit, the method of any one of the preceding Embodiments, comprising magnetic beads, actin, latrunculin, CaCh, ATP and DTT.

57. The composition, the kit, the method of any one of the preceding Embodiments, for use in stabilizing total cfDNA in a biological sample.

58. The composition, the kit, the method of any one of the preceding Embodiments, having a pH of above about 7.

59. The composition, the kit, the method of any one of the preceding Embodiments, having a pH of between about 7 and about 12.

60. The composition, the kit, the method of any one of the preceding Embodiments, having a pH of between about 7 and about 8.

61. The composition, the kit, the method of any one of the preceding Embodiments, comprising between about 0.05mM and about 5 mM CaCh.

62. The composition, the kit, the method of any one of the preceding Embodiments, comprising between about 0.05mM and about 1 mM CaCh.

63. The composition, the kit, the method of any one of the preceding Embodiments, comprising between about 0.05 mM ATP and abut 5 mM ATP.

64. The composition, the kit, the method of any one of the preceding Embodiments, comprising between about 0.05 mM ATP and abut 1 mM ATP.

65. The composition, the kit, the method of any one of the preceding Embodiments, comprising between about 0.05 mM DTT and about 5 mM DTT.

66. The composition, the kit, the method of any one of the preceding Embodiments, comprising between about 0.05 mM DTT and about 1 mM DTT.

67. The composition, the kit, the method of any one of the preceding Embodiments, comprising about O.lmM CaCh, about 0.2mM ATP, about 0.2mM DTT, about 3.3pg/ml actin, about 83pM latrunculin and about 15 pl magnetic beads solution. 68. The composition, the kit, the method of any one of the preceding Embodiments, comprising about 5mM Tris pH7.8, about O.lmM CaCh, about 0.2mM ATP, about 0.2mM DTT, about 3.3pg/ml actin, about 83pM latrunculin and about 15 pl magnetic beads solution.

69. The composition, the kit, the method of any one of the preceding Embodiments, for use in characterizing cfDNA in the urine sample.

70. The kit of any one of the preceding Embodiments optionally comprising instructions for preparing one or more urine samples for cfDNA characterization.

71. A kit comprising a first composition comprising at least one means of binding cfDNA and a second composition comprising one or more cfDNA stabilization reagents, the first composition and the second composition are each in a separate container means and optionally instructions for mixing the first composition with the second composition to obtain a composition comprising one or more means to bind cfDNA and/or one or more cfDNA stabilization reagents and optionally instructions for preparing one or more urine samples for cfDNA characterization.

72. The kit of any one of the preceding Embodiments comprising one or more filters.

73. The kit of any one of the preceding Embodiments comprising one or more urine collection means suitable for holding urine samples.

74. The kit of any one of the preceding Embodiments, wherein said instructions comprising collecting one or more urine samples in said urine collection means.

75. The kit the method of any one of the preceding Embodiments, wherein said instructions comprising filtering one or more urine samples to obtain one or more filtered urine samples.

76. The kit t of any one of the preceding Embodiments, wherein said instructions comprising contacting said urine sample or filtered urine samples with the composition.

77. The kit of any one of the preceding Embodiments, wherein said instructions comprises collecting at least one urine sample from a subject at a first time point and collecting at least one urine sample from said subject at a second time point, wherein said subject was administrated with at least one dose of a treatment between the first time point and the second time point.

78. The kit of any one of the preceding Embodiments, wherein said instructions comprises isolating a fraction of said one or more urine samples or said one or more filtered urine samples, wherein said fraction is suspected to comprise total cfDNA.

79. The method of any one of the preceding Embodiments, comprising filtering said one or more urine samples prior to said contacting.

80. The method of any one of the preceding Embodiments, comprising isolating a portion of the urine sample or the filtered urine sample, wherein said portion is suspected to comprise cfDNA.

81. The method of any one of the preceding Embodiments, comprising characterizing cfDNA in said portion.

82. The method of Embodiment 82, wherein said characterizing comprising contacting said portion with at least one cfDNA specific detecting molecules.

83. The method of any one of the preceding Embodiments, comprising collecting at least one urine sample from said subject at a first time point and at least one other urine sample from said subject at a second time point, wherein said subject received at least one dose of a treatment between said first time point and said second time point.

84. The method of Embodiment 83, wherein said first time point is before said subject received at least one dose of a treatment and said second time point is after said subject received at least one dose of a treatment.

85. The method of any one of the preceding Embodiments for assessing responsiveness of said subject to a treatment regimen by determining whether said subject is responsiveness to treatment regimen.

86. The method of any one of the preceding Embodiments comprising calculating the rate of change of said level of cfDNA between the at least one sample obtained at a time point prior to said treatment and the at least one sample obtained at a time point after initiation of said treatment; wherein a rate of change above a predetermined rate of change indicates that said subject belongs to a responsive population. 87. The method of Embodiment 86, wherein said predetermined rate of change is at least a three-fold increase.

88. The method of Embodiment 87, wherein the subject is classified as a responder if at least a three -fold increase in the TERT per ml urine is observed between the first time point and the at second time point, such that the subject received at least one dose of a treatment between the first time point and the second time point.

89. The method of any one of the preceding Embodiments, wherein said subject is suffering from a pathological disorder.

90. The method of Embodiment 89, wherein the pathological disorder is a proliferative disorder or a metastatic proliferative disorder.

91. The method of Embodiment 90, wherein said proliferative disorder is a carcinoma.

92. The method of Embodiment 91 , wherein said carcinoma is an adenocarcinoma, a basal cell carcinoma, or squamous cell carcinoma.

93. The method of Embodiment 92 wherein said proliferative disorder is at least one primary and/or secondary malignancy of at least one of breast cancer, bladder cancer, kidney cancer, hepatocarcinoma cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, melanoma, sarcoma, and specifically, osteosarcoma, ovarian cancer, prostate cancer, thyroid cancer, cervical cancer, uterus cancer, laryngeal cancer, brain cancer, hematopoietic malignancies (non-solid lymphoma, leukemia, or multiple myeloma).

94. The method of Embodiment 93, wherein the proliferative disorder is lung cancer (lung carcinoma).

95. The method of Embodiment 94, wherein said lung cancer is a non-small-cell lung cancer (NSCLC).

96. The method of any one of the preceding Embodiments, wherein said proliferative disorder is characterized by comprising at least one mutation in at least one protein kinase. 97. The method of Embodiment 96, wherein the protein kinase is a tyrosine kinase.

98. The method of Embodiment 96, wherein the at least one mutation in the protein kinase is at least one of growth factor receptor (GFR), c-Met, c-Kit, PI3-kinase, PI3CA, epidermal growth factor receptor (EGFR), Platelet-derived growth factor receptor (PDGFR), insulin receptor and insulin-like growth factor 1 receptor (IGF1R), stem cell factor (SCF) receptor, Anaplastic lymphoma kinase (ALK), fibroblast growth factor receptor 1 (FGFR1), Proto-oncogene tyrosine-protein kinase (ROS1), RET, NTRF1, HER2 (ERBB2), Tropomyosin receptor kinase A (TrkA), MEK1, MEK2, MAP2K1, ABL, Discoidin domain receptor family, member 1 (DDR1), Discoidin domain receptor family, member 2 (DDR2), quinone reductase 2 (NQ02), Vascular endothelial growth factor receptor (VEGFR), c-RAF, b-RAF, Granulocyte colony-stimulating factor (G- CSF), FET3, Src, Epidermal growth factor receptor (EGFR), lymphocyte-specific protein tyrosine kinase (Lek), EPHA3, EPHA8, Mitogen-activated protein kinase 11 (MAPK11), zipper containing kinase AZK (ZAK), Fibroblast Growth Factor Receptor (FGFR), ROS1, C-MET, JAK1, JAK2, AXL, flt3, BTK, IDH2, or combinations thereof.

99. The method of any one of the preceding Embodiments, wherein said treatment is or comprises at least one tyrosine kinase inhibitor (TKI).

100. The method of Embodiment 99, wherein said at least one TKI is at least one of Imatinib, Gefitinib, Erlotinib, Dasatinib, Sunitinib, Adavosertib, Lapatinib, Osimertinib, Crizotinib, Alectininb, Ceritinib, Brigatinib, Lorlatinib, Ensartinib, Sorafenib, Nilotinib, Pazopanib, Ruxolitinib, Vemurafenib, Vandetanib, Regorafenib, Cabozantinib, Axitinib, Bosutinib, Ponatinib, Afatinib, Trametinib, Dabrafenib, Ibrutinib, Ceritinib, Lenvatinib, Osimertinib, Alectinib, Cobimetinib, Neratinib, Brigatinib, Acalabrutinib, Midostaurin, Tivozanib, Enasidenib, Encorafenib, Dacomitinib, Lorlatinib, Binimetinib, Larotrectinib, Gilteritinib, erdafitinib, Pexidartinib, Entrectinib, Zanubrutinib, Fedratinib, Tucatinib, Avapritinib, Ripretinib, Pemigatinib, Capmatinib, Selpercatinib, Pralsetinib, Selumetinib, Infigratinib, Tepotinib, Mobocertinib, Asciminib or any combination thereof.

101. The method of Embodiment 100, wherein said at least one TKI is at least one of Gefitinib, Erlotinib, Sorafenib, Crizotinib, Afatinib, Trametinib, Dabrafenib, Ceritinib, Ensartinib, Osimertinib, Alectinib, Brigatinib, Dacomitinib, Lorlatinib, Entrectinib, Capmatinib, Selpercatinib, Pralsetinib, Tepotinib, Mobocertinib or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 is an exemplary kit for collecting multiple urine samples in accordance with some examples of the present disclosure; the exemplary kit comprise a plastic cap for urine collection, for example as the one denoted as “1”, one or more holders, for example a holder that is designed to hold and organize one or more urine collecting containers, for example as the ones denoted as “2” and “10” shown in this figure holding six tubes for six days (optionally consecutive) of urine collection and holding tubes after urine collection, a syringe, for example as the one denoted as “3”, a filter, for example a syringe filter (0.45 pm pore size) as the one denoted as “4” syringe, a holder for a single tube, for example the stand denoted as “5”, a vortex mixer, for example as the one denoted as “6” vortex, a magnet stand, for example as the one denoted as “7”, one or more liquid-handling instrument, for example, the pipette denoted as “8”, plastic pipette, one or more markers/pen denoted as “9”, marker pen, and disposable nitril gloves.

Figs. 2A-2C relate to evaluation of effect of Osimertinib (an EGFR inhibitor) treatment on epidermal growth factor receptor positive (EGFR⁺) non-small cell lung cancer (NSCLC) patient; Fig. 2A is a graph showing daily dynamics of total cfDNA levels in urine samples collected three days as baseline prior to treatment (denoted as -3, - 2 and -1) and four days after initiation of treatment (denoted as 1, 2, 3 and 4), Osimertinib treatment started on day -1 after the urine sample was collected (indicated by the arrow), Figs. 2B and 2C are positron emission tomography-computed tomography (PET-CT) recorded about one month before initiation of treatment and about 4 month after initiation of Osimertinib treatment, respectively, arrows in Fig. 2B indicate tumor/metastatic regions. Figs. 3A-3C relate to evaluation of effect of Osimertinib treatment on EGFR⁺ NSCLC patient; Fig. 3A is a graph showing daily dynamics of total cfDNA levels in urine samples collected one day prior to treatment initiation as baseline (denoted as -1) and four days after initiation of treatment (denoted as 1, 2, 3 and 4), Osimertinib treatment started on day -1 after the urine sample was collected (indicated by the arrow), samples were not taken for several days (red line indicates end of the 4 days) and followed by additional five samples from consecutive days(denoted as 9, 10, 11 and 12), Figs. 3B and 3C are PET-CT recorded about one month before initiation of treatment and about 2 month after initiation of Osimertinib treatment respectively; the circles region in Fig. 3B indicate tumor/metastatic regions.

Figs. 4A-4C relate to evaluation of effect of Osimertinib treatment after surgery on EGFR+ NSCLC patient; Fig.4A is a graph showing daily dynamics of total cfDNA levels in urine samples collected two days prior to treatment initiation as baseline (denoted as -2 and -1) and four days on treatment (denoted as 1, 2, 3 and 4), Osimertinib treatment started on day -1 after the urine sample was collected (indicated by the arrow), Figs. 4B and 4C are PET-CT recorded about 28 days before treatment and five months after initiation of Osimertinib treatment, respectively; the circles region in Fig. 4B indicate lytic region that as shown in Fig. 4C turned into sclerotic tissue.

Fig. 5 is a graph showing daily dynamics of total cfDNA levels in urine samples collected from an EML4-ALK positive NSCLC patient, samples were collected two days prior to treatment initiation as baseline (denoted as -2 and -1) and four days on treatment (denoted as 1, 2, 3 and 4) before and after brain irritation (indicated by the arrow).

Fig. 6 is a graph showing daily dynamics of total cfDNA levels from a healthy volunteer; urine samples were collected at indicated times.

DETAILED DESCRIPTION OF EMBODIMENTS

Determining treatment protocols suitable for an individual or a subset of individuals is highly desired as treatment protocols are often associated with different effects when tested on a large population of patients. In other words, while a given treatment protocol may be effective for one or more individuals, other individuals may experience treatment resistance as well as undesired side effects. Therefore, the ability to specifically select and tailor a treatment protocol before and/or at early stages after initiation of treatment and/or throughout or after a treatment period, may avoid inadequate treatments and improve changes of survival. In addition, predicting the chances of a specific patient or a subpopulation of patients to respond to treatment before initiation of the treatment or at early stages after initiation of the treatment is highly valuable and clinically desired.

The present disclosure generally relates to personalized medicine and is aimed at enabling decisions and practices to an individual patient by following changes in circulating free DNA (cfDNA) and/or circulating tumor DNA (ctDNA) in the patients.

Specifically, as described herein, the inventors developed compositions and kits that enable stabilization of cfDNA in biological samples for hours and even for days after samples collection and are effective in providing high quality cfDNA from the samples (such as from urine sample). Hence, the compositions and kits may be used as a unique platform allowing storage of biological samples (such as urine samples) before processing of the samples.

The stabilization of cfDNA as used herein can be understood as inhibition/reduction/preventing loss of cfDNA, optionally by degradation of cfDNA.

As shown herein, the application of the compositions and kits that stabilized cfDNA enabled detection of cfDNA in small amounts (nanogram, microgram) in the biological samples hours and days after being collected.

As shown below, the methods described herein making use of the compositions and kits are applicable to routine monitoring of cfDNA in the biological sample (such as urine sample) optionally on a daily basis (if and when required). This is highly advantageous as it offers a user-friendly and convenient solution for stabilization of cfDNA in home use.

As shown in the examples below, the inventors demonstrated that determining total cfDNA levels in biological sample, such as urine samples, provide information on treatment efficacy at early stages of treatment, for example several hours and days after initiation of treatment. It was suggested that the compositions, kits and methods may be suitable for determining (assessing) responsiveness to first-line treatment, second-line treatment or advanced lines treatment, preferably second line treatment.

Accordingly, in accordance with some aspects, the present disclosure provides a composition comprising one or more means to bind cfDNA and one or more cfDNA stabilization reagents. In some embodiments that may be considered as aspects of the present disclosure the composition is for use in stabilizing total cfDNA in a biological sample. In some embodiments that may be considered as aspects of the present disclosure the composition is for use in generating high-quality total cfDNA from a biological sample.

In some other aspects, it is provided a composition comprising one or more means to bind cfDNA and one or more cfDNA stabilization reagents suitable for use in stabilizing total cfDNA in a biological sample.

In some other aspects, it is provided use of one or more means to bind cfDNA and one or more cfDNA stabilization reagents for the preparation of a composition. In some examples and as noted above the composition may for use or may be suitable in stabilizing total cfDNA in a biological sample.

Biological sample as used herein refers to any sample obtained from the subject that comprise at least one cell or any fraction thereof. In some specific embodiments, sample applicable in the methods of the invention may be a body fluid. In some examples, the biological sample may be any one of blood, plasma, tissue extracts, urine, saliva. In some examples, the biological sample may be bone marrow, lymph fluid, blood cells, blood, serum, plasma, semen, spinal fluid or CSF, the external secretions of the skin.

In some examples, the biological sample is urine sample.

In some embodiments that may be considered as aspects of the present disclosure, the composition is for use in stabilizing total cfDNA in a urine sample. In some embodiments that may be considered as aspects of the present disclosure the composition is for use in generating high-quality total cfDNA from a urine sample. In some other aspects, it is provided a composition comprising one or more means to bind cfDNA and one or more cfDNA stabilization reagents suitable for use in stabilizing total cfDNA in a urine sample.

In some examples, the composition is for use in stabilizing cfDNA in a biological sample or a fraction thereof, such as a urine sample for a time period during which essentially no degradation of total cfDNA is detected. In some examples, the composition is for use in stabilizing cfDNA in a biological sample, such as a urine sample, at most 20 days, at times at most 15 days, at times at most 12 days, at times at most 10 days, at times at most 7 days, at times at most 5 days.

As shown herein, by combining means that enable immediate binding of cfDNA and stabilization of the bound cfDNA, the inventors were capable of preparing urine samples that can be either processed immediately or alternatively may be stored for hours or day such that regardless of the storage duration, accurate and reliable information on total cfDNA levels was obtained from these samples. These advantages make the compositions and kits described herein and methods of its use applicable for home use.

In the following text, when referring to the composition it is to be understood as also referring to the formulation, kit, method and use disclosed herein. Thus, whenever providing a feature with reference to the composition, it is to be understood as defining the same feature with respect to the formulation, kit, method or use mutatis mutandis.

As used herein the term cell-free DNA (cfDNA) refers to a DNA fragments that carries genome-wide DNA information and that was released from cells (i.e. exists outside a cell), usually after cell death. cfDNA is floating within a bodily fluid and may be found in extracellular vesicles or exosomes.

The cfDNA may be from a dead cell or a dying cell. In such case, DNA is fragmented and released from the cell.

The cfDNA can be obtained from any organism, for example, a human subject. In some embodiments, the cfDNA is a mammalian cfDNA. In some embodiments, the cfDNA is a. As appreciated, most of human cfDNA (about 70-90%) is derived from leukocytes, and other human cfDNA is derived from several other organs, such as the liver.

In some embodiments, the cfDNA is from a human genome.

The cfDNA in accordance with the present disclosure encompasses naked cfDNA or non-naked cfDNA. In addition, the cfDNA may be bound/associated/crosslinked with a protein.

In some embodiments, the cfDNA is fetal DNA. In some embodiments, the cfDNA is fetal cell free DNA (cfDNA).

In some embodiments, the cfDNA is viral DNA. In some embodiments, the cfDNA is bacterial DNA. In some embodiments, the cfDNA is fungal DNA. In some embodiments, the cfDNA parasitic DNA. In some embodiments, the cfDNA if from a pathogen.

In some embodiments, the cfDNA is a ctDNA.

Circulating tumor DNA (ctDNA) as used herein refers to DNA or fragments thereof that is found outside of a cell, for example, one that is in the bloodstream and enters kidney through bloodstream and encompasses to DNA that comes from cancerous cells and tumors.

As appreciated, large variabilities are usually observed in the % of ctDNA in cancer patients.

In some embodiments, ctDNA is derived from a primary tumor. In some embodiments, ctDNA is derived from metastases. In some embodiments, ctDNA is derived from circulating tumor cells (CTC).

As appreciated, as a tumor grows, cells die and are replaced by new ones and the dead cells undergo lysis and their contents, including DNA, are released into the bloodstream. Hence, ctDNA refers to small pieces of DNA (DNA fragments), usually comprising fewer than 200 building blocks (nucleotides) in length and at times having an average size of 167 bps. In other words, ctDNA is often considered as having a length of a DNA fragment wrapped around a histone core (the nucleosome, ±147 bps) and its Hl linker histone (±20 bps).

It is known that ctDNA can be also found in urine - a phenomenon known as “trans-renal”, in which DNA fragments are filtered through the kidney’s glomerular filtration system, into the urine. The resulting ctDNA fragments present in the urine are known as trans-renal ctDNA (TRctDNA).

In some embodiments, the cfDNA is extracted from bodily fluid. As noted herein, the cfDNA is extracted and obtained from at least one urine sample.

As known in the art, quantifying cfDNA levels in urine samples is challenging as urine includes a variety of normal cells (e.g. cells shedding from genitourinary tract, leukocytes) that may die and release their DNA. The level of such a non-relevant DNA increases with time and is collected with the cfDNA, masking the relevant cfDNA information that originated from the diseased cells. In addition, the DNA released from dead cells results in high background and variability between samples. In addition, urine samples comprise high levels of DNase that enhance degradation of cfDNA in the samples.

Yet, as exemplified herein, it was surprisingly found that it was possible to maintain stability of cfDNA for a time period of hours and even days. This was attributed to the unique developed composition.

In some embodiments, the composition comprises one or more means to bind cfDNA.

Means to bind cfDNA also denoted herein cfDNA binding means refers to any means that is capable and/or suitable of binding DNA e.g. cfDNA and/or ctDNA.

The binding of the DNA, preferably cfDNA to the cfDNA binding means may be direct binding, indirect binding, or both. In some examples in which the binding of the cfDNA to the cfDNA binding means is indirect binding, the cfDNA binding means may comprise a binding agent associated thereto. As appreciated, the binding agent is for use in facilitating and/or enabling the binding of the cfDNA. In some examples, the cfDNA binding means may encompass a solid support, at times denoted as solid phase support, solid phase carrier, solid carrier, carrier or the like.

The cfDNA binding means in accordance with the present disclosure is not limited to a specific solid support and is applicable to any solid support that can bind the cfDNA. In addition, the cfDNA binding means after being bound to cfDNA forms a complex, cfDNA binding means-cfDNA such that the complex may be separated from the biological sample (e.g. urine sample).

In some embodiments, the cfDNA binding means-cfDNA complex may be separated from the biological sample without use of centrifugation.

In some examples, the cfDNA binding means is a solid support.

In some embodiments, the solid support is or comprises an insoluble material.

In some embodiments, the solid support has a color that is different than the color of the biological sample.

In some examples, the solid support may be one or more of magnetic beads, super magnetic beads, glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, magnetite or a combination thereof.

The solid support may have different shapes (structural configuration). In some examples, the solid support may have a spherical shape. In some examples, the solid support may a bead. In some examples, the solid support may be a rod.

In some embodiments, the solid support is or comprises particles. In some embodiments, the solid support is or comprises nanoparticles.

As used herein the term nanoparticles refers to discrete particles, at least one of their dimensions being in the nanometric range, typically 2 nm to 500 nm in length or diameter. In some embodiments, the nanoparticles have an average diameter of about lOnm and about lOOnm, at times about lOnm and about 50nm.

The nanoparticle as used herein may refer to a population of nanoparticles that may be of a single type of nanoparticles or of a mixture of nanoparticle types. The various populations may be classified by the nanoparticle size, size distribution, shape, chemical composition.

In some embodiments, the cfDNA binding means is or comprises agarose gel.

In some embodiments, the cfDNA binding means is or comprises Sepharose beads.

In some embodiments, the cfDNA binding means is or comprises silica gel.

In some embodiments, the cfDNA binding means is or comprises polystyrene microspheres.

In some embodiments, the cfDNA binding means is or comprises magnetic beads. In some embodiments, the cfDNA binding means is or comprises super magnetic beads.

In some embodiments, the cfDNA binding means is or comprises magnetic nanoparticles.

In some embodiments, the magnetic beads comprise nanoparticles of iron oxides. In some embodiments, the magnetic beads comprise nanoparticles of magnetite (Fe3O4).

It was suggested that in examples in which the composition comprises a solid support, for example being or comprising magnetic beads, the solid support (e.g. magnetic beads) form a complex with the cfDNA (if present in the sample) allowing the separation/isolation of a fraction comprising the cfDNA from the biological sample, that in some examples is a urine sample.

It was suggested that in examples in which the solid support is or comprises magnetic nanoparticles, the magnetic nanoparticles form a complex with the cfDNA (if present in the sample) allowing the separation/isolation of a fraction comprising the cfDNA from the biological sample, that in some examples is a urine sample.

In some embodiments, the composition comprises magnetic beads in excess in order to allow binding of cfDNA from the sample.

In some embodiments, the composition comprises between about 2pl magnetic beads and about 30pl magnetic beads. In some embodiments, the composition comprises between about 5pl magnetic beads and about 25pl magnetic beads. In some embodiments, the composition comprises between about lOpl magnetic beads and about 30pl magnetic beads. In some embodiments, the composition comprises between about lO l magnetic beads and about 20pl magnetic beads. In some embodiments, the composition comprises about lOpl magnetic beads. In some embodiments, the composition comprises about 15pl magnetic beads. In some embodiments, the composition comprises about 20pl magnetic beads.

In some embodiments, the composition comprises one or more cfDNA stabilization reagent (or agent).

As used herein the term cfDNA stabilizing reagent refers to one or more reagents that collectively may directly or indirectly protect cfDNA and hence prevent, inhibit, reduce cfDNA damage or degradation. Damage or degradation of cfDNA typically occurs during storage or transportation.

The present disclosure is not limited to a specific cfDNA stabilizing reagent and is applicable to a variety of reagents provided that collectively are capable of stabilizing cfDNA in the sample (protect cfDNA from damage or degradation) on one hand and on the other hand do not interfere with the binding of the cfDNA to the cfDNA binding means.

As appreciated, stabilization of cfDNA by reducing cfDNA degradation allows storage of the samples (for example urine samples) for a time period of hours or at times of days. As shown below, this enables to obtain accurate and reliable information on the cfDNA in the sample.

In some examples, the cfDNA stabilizing reagent is a chelating agent or an inhibitor.

In some examples, the cfDNA stabilizing reagent is or comprises a protease inhibitor, a nucleases inhibitor or a combination thereof.

In some examples, the one or more cfDNA stabilizing reagents may inhibit DNase activity. The one or more cfDNA stabilizing reagents may include one or more reagents that collectively inhibit DNase activity and hence inhibit degradation of cfDNA in the sample.

In some embodiments, the one or more cfDNA stabilizing reagents is or comprises at least one DNase inhibitor.

DNase inhibitor as used herein refer to a substance that may prevent or inhibit the activity of DNases that are enzymes which specifically degrade DNA.

In some embodiments the DNase inhibitor is selected such that it does not interfere with the binding of the cfDNA to the cfDNA binding means.

In some embodiments, the DNase inhibitor is a natural DNase inhibitor, a synthetic DNase inhibitor, or any combination thereof.

In some embodiments, the DNase inhibitor is a DNase I inhibitor, a DNase II inhibitor, or any combination thereof.

In some embodiments, the DNase inhibitor is a DNase I inhibitor.

In some embodiments, the natural DNase inhibitor can be isolated from one ore more of a human source, an animal source, a microorganism source, a plant source or any combination thereof.

In some embodiments, the natural DNase inhibitor is a microorganism source DNase. In some embodiments, the microorganism source DNase inhibitor is one or more antibiotics isolated from the genus Streptomyces. In some embodiments, the microorganism source DNase inhibitor is one or more of actinomycin D, nogalamycin, daunomycin, neomycin B, paromomycin or any combination thereof.

In some embodiments, the natural DNase inhibitor is an animal source DNase. In some embodiments, the animal source DNase is at least one of actin, anti-DNase antisera or any combination thereof. In some embodiments, the at least one DNase inhibitor is actin. In some embodiments, the DNase inhibitor is a synthetic DNase I inhibitor, a synthetic DNase y inhibitor, a synthetic DNase II inhibitor, a synthetic DFF40/CAD inhibitor, or any combination thereof.

In some embodiments, the DNase inhibitor is an inorganic DNase inhibitor.

As noted herein, the at least one DNase inhibitor is actin.

In some embodiments, the composition comprises between about 50 ng/ml and about 20 pg/ml actin. In some embodiments, the composition comprises between about 75 ng/ml and about 15 pg/ml actin, at times between about 0.1 pg/ml and about 15 pg/ml actin, at times between about 0.15pg/ml and about 15 pg/ml actin, at times between about 0.2pg/ml and about 15 pg/ml actin, at times between about 0.2pg/ml and about 10 pg/ml actin, at times between about 0.2pg/ml and about 10 pg/ml actin, at times between about 0.5pg/ml and about 10 pg/ml actin, at times between about 0.5pg/ml actin and about 8 pg/ml actin, at times between about 1 pg/ml actin and about 6 pg/ml actin, at times between about 2pg/ml actin and about 5 pg/ml actin. In some embodiments, the composition comprises about 2 pg/ml. In some embodiments, the composition comprises about 3.3pg/ml actin. In some embodiments, the composition comprises about 4pg/ml actin. In some embodiments, the composition comprises about 5pg/ml actin.

As used herein the term actin refers to a family of globular multi-functional proteins that form microfilaments in the cytoskeleton, and the thin filaments in muscle fibrils. Actin can be present as a free monomer denoted as G-actin (globular) or as part of a linear polymer microfilament denoted as F-actin (filamentous).

In some examples in which the cfDNA stabilizing reagents comprises a DNase inhibitor being actin, the cfDNA stabilizing reagents may comprise means to maintain actin in a monomeric form. In other words, the one or more cfDNA stabilizing reagents comprises actin and one or more agents that maintain actin in a form (e.g. monomeric form) that may bind DNase and inhibit its activity.

Without being bound by theory, it was suggested that maintaining actin in a monomer form by preventing/inhibiting/reducing actin polymerization would inhibit DNase activity, stabilize cfDNA in the urine samples and allow storage of the urine samples.

The term actin filament inhibitor is used to denote an agent that maintain actin in monomer form and hence inhibit, reduce, prevent actin assembly into filament structure.

The present disclosure is not limited to a specific actin filament inhibitor.

In some embodiments, the cfDNA stabilizing reagent is or comprises an actin filament inhibitor.

In some examples, the actin filament inhibitor is at least one of substoichiometric cytochalasin B (CB), cytochalasin D, latrunculin or a combination thereof.

In some embodiments, the actin filament inhibitor is latrunculin.

In some embodiments, the composition comprises between about I M and about 150pM latrunculin, at times between about 5pM and about 150pM latrunculin, at times between about lOpM and about 150pM latrunculin, at times between about 15pM and about 150pM latrunculin, at times between about 20pM and about 150pM latrunculin, at times between about 25pM and about 150pM latrunculin, at times between about 30pM and about 150pM latrunculin, at times between about 35pM and about 150pM latrunculin, at times between about 40pM and about 150 pM latrunculin, at times between about 45pM and about 150pM latrunculin, at times between about 50 pM latrunculin and about 150pM latrunculin. In some embodiments, the composition comprises between about 50pM latrunculin and about lOOpM latrunculin. In some embodiments, the composition comprises between about 70pM latrunculin and about lOOpM latrunculin. In some embodiments, the composition comprises about 70pM latrunculin. In some embodiments, the composition comprises about 75pM latrunculin. In some embodiments, the composition comprises about 80pM latrunculin. In some embodiments, the composition comprises about 83pM latrunculin. In some embodiments, the composition comprises about 85pM latrunculin. In some embodiments, the composition comprises about 90pM latrunculin. In some embodiments, the composition comprises about lOOpM latrunculin. As used herein the term latrunculin refers to a family of natural products and toxins produced by sponges, including genus Latrunculia and Negombata that may bind actin monomers and reduces/prevents/inhibit actin polymerization.

In some embodiments, the one or more cfDNA stabilizing reagents comprises latrunculin A, latrunculin B or any combination thereof.

In some embodiments, the composition comprising at least one DNase inhibitor and at least one actin filament inhibitor.

In some embodiments, the one or more cfDNA stabilizing reagents comprises actin and one or more actin filament inhibitor.

In some embodiments, the composition comprising at least one DNase inhibitor and latrunculin.

In some embodiments, the one or more cfDNA stabilizing reagents comprises actin and latrunculin.

The composition described herein may be provided in any form, including, inter alia, a solution, a lyophilization product or the like. As appreciated, the composition present as a lyophilized product may be reconstituted in an aqueous solution or a formulation as further described below.

In some examples, the composition comprised at least one cfDNA binding means and actin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means and one or more actin filament inhibitor. In some examples, the composition comprised at least one cfDNA binding means and latrunculin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, at least one DNase inhibitor and one or more actin filament inhibitor. In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, actin and one or more actin filament inhibitor.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, at least one DNase inhibitor and latrunculin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, actin and latrunculin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads and at least one DNase inhibitor. In some examples, the composition comprised magnetic beads and actin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles and at least one DNase inhibitor. In some examples, the composition comprised magnetic nanoparticles and actin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads and one or more actin filament inhibitor. In some examples, the composition comprised magnetic beads and latrunculin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles and one or more actin filament inhibitor. In some examples, the composition comprised magnetic nanoparticles and latrunculin. In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, at least one DNase inhibitor and one or more actin filament inhibitor.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads, at least one DNase inhibitor and one or more actin filament inhibitor.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles, at least one DNase inhibitor and one or more actin filament inhibitor.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, actin and one or more actin filament inhibitor.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads, actin and one or more actin filament inhibitor.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles, actin and one or more actin filament inhibitor.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads, at least one DNase inhibitor and latrunculin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles, at least one DNase inhibitor and latrunculin. In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, actin and latrunculin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads, actin and latrunculin.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles, actin and latrunculin.

In some embodiments, the composition has a pH of above about 7, at times above about 7.2, at times above about 7.5, at times above about 7.7.

In some embodiments, the composition has a pH of between about 7 and about 12, at times between about 7 and about 10, at times between about 7 and about 9, at times between about 7 and about 8.5, at times between about 7 and about 8.

In some embodiments, the composition has a pH of about 7, at times about 7.2, at times about 7.4, at times about 7.5, at times about 7.6, at times about 7.7, at times about 7.8, at times about 8.

In some embodiments, the composition comprises between about ImM and about lOmM Tris pH 7.8. In some embodiments, the composition comprises about 5mM Tris pH 7.8.

It should be understood that in addition to the components mentioned above, the composition may also include other components. In some embodiments, the composition may optionally further comprise at least one of pharmaceutically acceptable carrier/s, excipient/s, additive/s diluent/s and adjuvant/s.

Additionally or alternatively, the composition may comprises additional components that may assist in maintaining the stability of the cfDNA. In some examples, the composition may comprise one or more components that maintain actin in a monomeric form. In some embodiments, the composition comprising at least one salt. The present disclosure is not limited to a specific salt provided that the at least one salt is capable of maintaining actin in a monomeric form, i.e. prevents/reduces actin polymerization.

In some embodiments, the composition comprises CaCh.

In some embodiments, the composition comprises between about 0.05mM and about 5mM CaCh, at times between about 0.05mM and about 3mM CaCh, at times between about 0.05mM and about ImM CaCh, at times between about 0.05mM and about 0.5mM CaCh, at times between 0.05 mM and about 0.3 mM CaCh. In some embodiments, the composition comprises about 0.05mM CaCh. In some embodiments, the composition comprises about O.lmM CaCh. In some embodiments, the composition comprises about 0.2mM CaCh.

In some embodiments, the composition comprises at least one nucleotide.

In some embodiments, the composition comprises one or more of adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP) or a combination thereof.

In some embodiments, the composition comprises ATP.

In some embodiments, the composition comprises between about 0.05mM ATP and about 5mM ATP, at times between about 0.05mM ATP and about 3mM ATP, at times between about 0.05 mM ATP and about 1 mM ATP, at times between about 0.05 mM ATP and about 0.5 mM ATP, at times between about 0.1 mM ATP and about 0.5 mM ATP, at times between about 0.1 mM ATP and about 0.3 mM ATP. In some embodiments, the composition comprises about O.lmM ATP. In some embodiments, the composition comprises about 0.2mM ATP. In some embodiments, the composition comprises about 0.3mM ATP.

In some embodiments, the composition comprises a sulfur containing compound.

In some embodiments, the composition comprises a reducing agent.

In some embodiments, the composition comprises a dithiol containing compound, a diol containing compound or a combination thereof. In some embodiments, the composition comprises Dithiothreitol (DTT).

In some embodiments, the composition comprises between about 0.05mM DTT and about 5mM DTT, at times between 0.05mM DTT and about 3mM DTT, at times between about 0.05 mM DTT and about 1 mM DTT, at times between about 0.05 mM DTT and abut 0.5 mM DTT, at times between about 0.1 mM DTT and abut 0.5 mM DTT, at times between about 0.1 mM DTT and abut 0.3 mM DTT. In some embodiments, the composition comprises about O.lmM DTT. In some embodiments, the composition comprises about 0.2mM DTT. In some embodiments, the composition comprises about 0.3mM DTT.

In some embodiments, the composition comprising one or more of (i) a salt, (ii) a nucleotide, (iii) sulfur containing compound or (iv) a combination thereof.

In some embodiments, the composition comprising one or more of (i) a salt, (ii) a nucleotide, (iii) a reducing agent or (iv) a combination thereof.

In some embodiments, the composition comprising one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, at least one DNase inhibitor, one or more actin filament inhibitor and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, at least one DNase inhibitor, one or more actin filament inhibitor and CaCh.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, at least one DNase inhibitor, one or more actin filament inhibitor and ATP.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, at least one DNase inhibitor, one or more actin filament inhibitor and DTT. In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads, at least one DNase inhibitor, one or more actin filament inhibitor and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles, at least one DNase inhibitor, one or more actin filament inhibitor and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, actin, one or more actin filament inhibitor and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads, actin, one or more actin filament inhibitor and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles, actin, one or more actin filament inhibitor and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, at least one DNase inhibitor, latrunculin and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads, at least one DNase inhibitor, latrunculin and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof. In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles, at least one DNase inhibitor, latrunculin and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising at least one cfDNA binding means, actin, latrunculin and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic beads, actin, latrunculin and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In accordance with some embodiments that may be considered as aspects of the present disclosure, the composition comprising magnetic nanoparticles, actin, latrunculin and one or more of (i) CaCh, (ii) ATP, (iii) DTT or (iv) a combination thereof.

In some embodiments, the composition comprising magnetic beads, actin, latrunculin and CaCh. In some embodiments, the composition comprising magnetic nanoparticles, actin, latrunculin and CaCh.

In some embodiments, the composition comprising magnetic beads, actin, latrunculin and ATP. In some embodiments, the composition comprising magnetic nanoparticles, actin, latrunculin and ATP.

In some embodiments, the composition comprising magnetic beads, actin, latrunculin and DTT. In some embodiments, the composition comprising magnetic nanoparticles, actin, latrunculin and DTT.

In some embodiments, the composition comprising actin, latrunculin, magnetic beads, CaCh, ATP and DTT.

In some embodiments, the composition comprising about 0.1 mM CaCh, about 0.2mM ATP, about 0.2mM DTT, about 3.3pg/ml actin, about 83pM latrunculin and about 15 pl magnetic beads solution. In some embodiments, the composition comprising about 5mM Tris pH7.8, about O.lmM CaCh, about 0.2mM ATP, about 0.2mM DTT, about 3.3pg/ml actin, about 83pM latrunculin and about 15 pl magnetic beads solution.

In some embodiments, the composition consisting of actin, latrunculin, magnetic beads, CaCh, ATP and DTT.

The composition described herein may be provided in a form of a kit optionally comprising instructions for use as described herein below.

In some embodiments, the kit is provided with instructions for collecting one or more, at times two or more urine samples from a subject.

Hence, in accordance with some aspects, the present disclosure provides a kit.

In some embodiments, the kit of the invention is suitable for collecting one or more, at times two or more urine samples from a subject and preparing cfDNA samples therefrom. In some examples, the kit is a urine collection kit.

In some embodiments, the kit comprises the composition described herein. In some examples, the kit is for use in preparing urine samples collected from a subject for characterizing cfDNA, if present in the samples.

In some embodiments, the kit comprises one or more urine collection means suitable for holding urine samples collected from the subject.

In some embodiments, the kit comprises instructions for use the kit.

In some embodiments, the instructions comprise instructions for filtering the one or more urine samples collected from the subject. In some embodiments, the kit comprises one or more filters suitable for filtering urine samples. In some embodiments, the filter has a size of about 0.45 micron (pm). It should be noted that the filter size is also denoted as pore size. As appreciated, using a filter with a filter size of about 0.45 micron would allow to separate out all urine components that are larger than 0.45 micron and collect the filtered urine sample.

In some embodiments, the instructions comprise instructions for adding a urine sample or a filtered urine sample to the composition. In some embodiments, the kit comprises the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents in a reservoir. In some embodiments in which the composition is provided in a reservoir, the kit comprises container means suitable for holding the composition and into which the reservoir of the composition may be partitioned. It should be noted that the number of the containers into which the reservoir is to be partitioned is similar or larger than the number of urine samples to be collected from the subject.

In some embodiments, the kit comprises one or more container means each container means comprising the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents. It should be noted that at times each container may comprise different amounts of the composition. It should be further noted that the number of the containers comprising the composition is similar or larger than the number of urine samples to be collected from the subject.

In some embodiments, the kit comprises a first composition comprising at least one means of binding cfDNA and a second composition comprising one or more cfDNA stabilization reagents. In some embodiments, the first composition and the second composition are each in a separate container means. In some embodiments, the kit may comprise a first composition comprising magnetic beads and a second composition comprising actin and latrunculin. In some embodiments, the kit may comprise a first composition comprising magnetic nanoparticles and a second composition comprising actin and latrunculin.

In such embodiments, in which the kit comprises first and second composition, the kit may comprise instructions for mixing the first composition with the second composition. This is done in order to obtain (receive) the composition described herein. In such cases, the mixing may be done by adding any one of the compositions into one of the container means of the other composition or alternatively mixing the compositions in an empty container means. In such examples, the kit comprises one or more empty container means suitable for mixing of the compositions. In some embodiments, each one of the first composition or the second composition may be added sequentially to the urine sample or the filtered urine sample. In some embodiments, the first composition is added to the urine sample or the filtered urine sample prior to the second composition. In some embodiments, the first composition is added to the urine sample or the filtered urine sample after the second composition.

In some embodiments, the kit may comprise one or more additional compositions comprising one of pharmaceutically acceptable carrier/s, excipient/s, additive/s diluent/s and adjuvant/s.

In some embodiments, the kit may comprise one or more additional compositions comprising at least one of CaCh, ATP, DTT or any combination thereof. Each one of or two or more of CaCh, ATP, DTT may be present in a different container means in the kit or all of CaCh, ATP, DTT may be present in a single container in the kit. The instructions may include also mixing such components into the composition (in case absent).

In some embodiments, the instructions comprise instructions for isolating a fraction suspected of comprising cfDNA from the compositions comprising the urine sample or the filtered urine samples. In some embodiments, the instructions comprising use of a magnet to isolate the fraction. In such embodiments, the kit comprises one or more magnets.

In some embodiments, the kit comprises at least one or more holders designed to hold and organize one or more container means suitable for holding the composition. In some examples, the holder is designated to hold one or more Eppendorf tubes that may be used as the container means.

In some embodiments, the kit comprises one or more syringes.

In some embodiments, the kit comprises vortex mixer.

In some embodiments, the kit comprises a magnet stand.

In some embodiments, the kit comprises one or more liquid-handling instrument. In some embodiments, the liquid-handling instrument is a plastic pipette.

An exemplary kit is shown in Fig. 1. In some examples, the kit is presented in Figure 1. In some embodiments, the kit comprises means to obtain information from the isolated cfDNA fraction (if present in the urine sample).

In some embodiments, the kit comprises one or more cfDNA specific detecting molecule.

In some embodiments the cfDNA specific detecting molecule may be selected from detecting nucleic acid molecules, detecting amino acid molecules or any combination thereof.

In some embodiments the nucleic acid detecting molecules comprise isolated oligonucleotides, each oligonucleotide specifically hybridizes to a nucleic acid sequence and optionally, to a control reference cfDNA.

In some embodiments the detecting molecules may be a pair of primers.

The term primer as used herein refers to an oligonucleotide (naturally occurring or synthetic) that provides a starting point for DNA synthesis and can be used to perform the polymerase chain reaction to copy pieces of DNA or for DNA sequencing.

In some embodiments, the primer used by the methods and kits of the present disclosure may comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides or more.

In some exemplary embodiments, the primers may be as follows:

F primer TERT F_TERT_3815 5' CCC TCC TTT GCC TTC CAC 3'

R primer TERT R_TERT_3875 5' GGT CAC TCC AAA TTC CCA

GA 3.

In some embodiments in which the cfDNA specific detecting molecule are oligonucleotides, specifically a pair of primers, the level of the cfDNA may be determined using a nucleic acid amplification assay as described herein below.

The instructions in the kit may include any of the method steps described below in connection with the methods of the invention.

Hence, in accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample for characterizing cell free DNA (cfDNA) in the at least one sample. As described herein, the information obtained this characterization may be useful in assessing responsiveness to treatment and/or to monitor disease progression.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises contacting the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, the one or more urine samples have been collected from a subject.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises collecting one or more urine samples from a subject and contacting each one of the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents.

In some embodiments, the at least one urine samples are collected from a subject. As appreciated, a subject in need thereof can independently collect the one or more urine samples.

In order to minimize variations in the measurements, it was suggested that the one or more urine samples preferably should have substantially the same pH value. In some examples, the one or more urine samples may be collected after the first urine of the day. In some examples, the one or more urine samples may be collected at the second urine of the day. It was also suggested that in order to maintain a substantially similar pH value in all collected samples, the subject should maintain a similar or even identical diet in the collection days.

In some embodiments, the methods comprise filtering the at least one urine samples prior to said contacting.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises the steps of filtering at least one urine sample to obtain a filtered urine sample and contacting the at least one filtered urine sample with the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, the one or more urine samples have been collected from a subject.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises the steps of collecting one or more urine samples from a subject, filtering the one or more urine samples to obtain a filtered urine sample and contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents.

The conditions (e.g. time) of the filtration step are selected to allow removal of intact cells and preventing cell death in the urine sample.

It was suggested that the filtration removes intact cells from the sample. It was further suggested that the filtration step should be done shortly after the one or more urine sample is collected in order to avoid/minimize/reduce death of cells, including, inter alia, cells from the genitourinary tract and/or leukocytes.

Hence, the time of the filtration step is adjusted to allow removal of intact cells.

In some embodiments, the filtration step is performed at a time in which the sample comprises intact cells.

In some embodiments, the filtration step is performed at most about 10 minutes after a urine sample is collected, at times at most about 8 minutes, at times at most about 7 minutes, at times at most about 5 minutes, at times at most about 4 minutes after a urine sample is collected.

In some embodiments, the filtration step is performed immediately after the urine sample is collected.

In some embodiments, the method comprises filtrating a urine sample at time of between about 30 seconds to about 10 minutes after being collected, at times between about 30 seconds to about 8 minutes, at times between about 30 seconds to about 5 minutes, at times between about 1 seconds to about 8 minutes, at times between about 30 seconds to about 5 minutes after the urine sample is collected. In some embodiments, the filtration step removes from the urine sample components that are larger than about 0.3 micron, at times about 0.35 micron, at times about 0.4 micron, at times about 0.45 micron. In some embodiments, filtration is configured to remove cells from the urine sample.

As described herein, the methods of the invetion comprising a contacting step of the urine sample or the fitlered urine sample with the compistion described herein.

The term contacting as used herein means to bring, put, incubates or mix together. In the context of contacting the urine sample or the fileted urine sample with the composition comprising cfDNA binding means and/or one or more cfDNA stabilizing reagents, the term "contacting" includes all measures or steps which allow interaction between the urine sample or any fraction thereof, with cfDNA binding means and/or the one or more cfDNA stabilizing reagents as described herein.

For example, the contacting is performed in a manner so that the urine sample or the filtered urine sample can interact with or bind to the cfDNA binding means and/or one or more cfDNA stabilizing reagents. The urine sample or the filtered urine sample can be contacted with the cfDNA binding means and the one or more cfDNA stabilizing reagents together (stimulatingly) or alternatively, the urine sample or any fraction thereof can be incubated with the cfDNA binding means and thereafter with the one or more cfDNA stabilizing reagents.

The binding between the cfDNA and the cfDNA binding means (for example magnetic beads or magnetic nanoparticles) may be covalent binding or non-covalent, reversible binding, e.g., binding via salt bridges, hydrogen bonds, hydrophobic interactions, or a combination thereof.

In some embodiments, the binding between the cfDNA and the cfDNA binding means (for example magnetic beads or magnetic nanoparticles) may be non-covalent binding.

In some embodiments, the binding between the cfDNA and the cfDNA binding means (for example magnetic beads or magnetic nanoparticles) may be covalent binding. The contacting is done under conditions (time or temperature) to allow binding of the urine sample or any fraction thereof to the cfDNA binding means and/or the one or more cfDNA stabilizing reagents.

In some examples, in which the method comprises contacting one or more urine samples with the composition without prior filtering, the methods may comprise collecting one or more urine samples directly into a composition comprising one or more cfDNA binding means and/or one or more cfDNA stabilizing reagents.

In some embodiments, the composition used by the methods comprise one or more cfDNA binding means and/or one or more cfDNA stabilizing reagents and one or more of CaCh.

In some embodiments, the composition used by the methods comprise magnetic beads, actin and latrunculin and CaCh.

In some embodiments, the composition used by the methods comprise one or more cfDNA binding means and/or one or more cfDNA stabilizing reagents and ATP.

In some embodiments, the composition used by the methods comprise magnetic beads, actin latrunculin and ATP.

In some embodiments, the composition used by the methods comprise one or more cfDNA binding means and/or one or more cfDNA stabilizing reagents and DTT.

In some embodiments, the composition used by the methods comprise magnetic beads, actin latrunculin and DTT.

In some embodiments, the composition used by the methods comprise one or more cfDNA binding means and/or one or more cfDNA stabilizing reagents, CaCh, ATP and DTT.

In some embodiments, the composition used by the methods comprise magnetic beads, actin, latrunculin, CaCh, ATP and DTT.

In some embodiments, the composition used by the methods comprise 5mM Tris pH7.8, O.lmM CaCh, 0.2mM ATP, 0.2mM DTT, 3.3pg/ml actin, 83pM latrunculin and 15 pl magnetic beads solution. In some embodiments, the method comprises isolating a portion of the urine sample or the filtered urine sample, wherein the portion is suspected to comprise cfDNA.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises contacting the at least one urine sample collected from a subject with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, and isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises collecting one or more urine samples from a subject, contacting each one of the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents and isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises the steps of filtering at least one urine sample collected from a subject to obtain a filtered urine sample, contacting the at least one filtered urine sample with the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, and isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises the steps of collecting one or more urine samples from a subject, filtering the one or more urine samples to obtained a filtered urine sample, contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents and isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA.

Isolating the portion of the urine sample can be done by any known means in the art. For example, isolating can be done by subjecting the urine sample or the filtered urine sample to a magnet for a sufficient time to allow isolation of a portion of the sample. The methods of the present disclosure comprise characterizing cfDNA in at least one urine samples. As described herein, the methods are for characterizing cfDNA in the one or more urine samples.

In some embodiments, the methods comprise characterizing cfDNA in the one or more urine samples, the filtered urine sample or the portion thereof suspected of comprising cfDNA.

Hence, in accordance with some aspects, the present disclosure provides a method for characterizing cfDNA in at least one urine samples collected from a subject and prepared as described herein.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises contacting the at least one urine sample collected from a subject with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises collecting one or more urine samples from a subject, contacting each one of the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises the steps of filtering at least one urine sample collected from a subject to obtain a filtered urine sample, contacting the at least one filtered urine sample with the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA. In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample, the method comprises the steps of collecting one or more urine samples from a subject, filtering the one or more urine samples to obtained a filtered urine sample, contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA.

The term characterizing cfDNA as used herein refers to obtaining/determining information related to cfDNA that is useful, for example, in determining treatment for a subject in need thereof.

In some embodiments, characterizing cfDNA comprises determining one or more of total cfDNA level, cfDNA concentration, cfDNA size, nucleosomes position, epigenetic marks or any combination thereof.

In some embodiments, characterizing cfDNA comprises determining number of the copies of Telomerase reverse transcriptase (TERT) per ml urine in one or more urine samples.

In some embodiments, characterizing cfDNA comprises determining cfDNA size distribution in one or more urine samples.

In some embodiments, characterizing cfDNA comprises determining nucleosomes positions in cfDNA in one or more urine samples.

The term nucleosomes positioning as used herein refer to an indication regarding the location of nucleosomes with respect to the genomic DNA sequence.

In some embodiments, characterizing cfDNA comprises determining epigenetic marks in cfDNA in one or more urine samples.

The term epigenetic marks as used herein refers to changes to DNA or its packaging components that alter gene expression, effectively turning gene transcription on and off, and that are inherited by daughter cells. Non-limiting examples of epigenetic modifications include CpG dinucleotide methylation. In some embodiments, characterizing cfDNA comprises determining total cfDNA level in one or more urine samples.

In some embodiments, characterizing cfDNA comprises determining total cfDNA level in one or more urine samples using real time PCR.

In some embodiments, characterizing cfDNA comprises cfDNA or ctDNA sequencing. In some embodiments, characterizing cfDNA is by next-generation sequencing (NGS). In some embodiments, characterizing cfDNA is by whole-exome sequencing (WES).

As appreciated, characterizing cfDNA by sequencing, such as NGS, may be used to obtain specific information on the frequency of mutations within a sample, specifically frequency or abundance of mutations in the cfDNA (or ctDNA).

In some embodiments, characterizing cfDNA comprises determining cfDNA methylation.

DNA methylation used herein refers to an epigenetic modification involving addition of a methyl group to a DNA molecule.

It should be noted that in accordance with some embodiments of the present disclosure, determining total cfDNA level involves determining total cfDNA level in the one or more urine samples or filtered urine samples.

As appreciated, the ability to obtain reliable and consistent determination of total cfDNA level in the one or more biological samples, such as urine samples may provide a broad measure that is independent from a specific genetic alternation. In other words, the ability to determination of total cfDNA level as shown herein is highly important as it does not require a priori knowledge of the tumor’s mutation(s). It was suggested that the information obtained from the total cfDNA level may be more applicable for second line treatment and advanced lines treatment, specifically in cases in which the treatment is selected based on factors other than somatic mutations. cfDNA characterization may be done by any method known in the art. In some examples, cfDNA level may be determined using at least one cfDNA specific detecting molecule. In some embodiments, the nucleic acid amplification assay includes one or more of a Real-Time PCR, micro array, PCR, in situ hybridization and comparative genomic hybridization.

In some embodiments, determining cfDNA level in the at least one urine sample can be done using a home read-out.

As shown herein below, the inventors demonstrated a correlation between an increase in the total cfDNA level and responsiveness to treatment. Specifically, an increase in transient cfDNA peak was detected after treatment initiation in patients and this increase was associated with responsiveness to treatment as concluded by radiographic assessment 2-4 months after treatment initiation.

As shown in Example 2 below, prospective data were obtained from epidermal growth factor receptor (EGFR)-positive non-small cell lung cancer (NSCLC) patients treated with Osimertinib, a first-line therapy for treatment of NSCLC, that is associated with superior disease-free survival (DFS) rates.

Specifically, Examples 2A-2C demonstrate an increase of about 6-fold to 16-fold, in transient cfDNA peak immediately after treatment initiation (between about 12 and 72 hours) in the patients diagnosed with NSCLC and this increase in transient cfDNA peaks was associated with responsiveness to treatment as concluded by radiographic assessment 2-4 months after treatment initiation.

Further as shown in Example 2D, a 4-fold increase in transient cfDNA peak was observed immediately after brain irradiation in patients with brain metastasis and this increase in transient cfDNA peak was associated with responsiveness to treatment.

The results shown in Example 2E demonstrate the specificity of the increase as no change in the cfDNA level with time was observed in samples collected from a healthy control volunteer.

Hence, the results suggest the observed increase in cfDNA can be considered as indicative to responsiveness to treatment in primary tumors as well as in metastasis.

Based on the above, the inventors envisaged that the compositions and kits described herein may be used in methods for continuous/dynamic monitoring of total cfDNA levels or ctDNA by NGS in biological samples such as urine samples collected from a subject at different time points. The urine samples may be processed immediately after being collected or stored for future processing, for example, after all samples were collected.

As described herein, the methods making use of the kit may be applicable for preparing one or more urine samples, the one or more urine samples may be collected from a subject before initiation of treatment, during treatment or at any time after treatment has been finished.

The composition, kit and method described herein may have a valuable potential to serve as a non-invasive tool for early prediction of treatment response, even within hours or days after initiation of treatment. It was suggested it may reduce routine radiology scans and thereby enable clinicians to optimize personalized therapy using easy to use non-invasive means.

In accordance with some aspects, the present disclosure provides a method of preparing at least one urine sample such that the at least one urine sample is collected from a subject who received at least one dose of a treatment.

The methods of the invention comprise collecting (obtaining) at least one urine sample and at times at least two urine samples in order to characterize cfDNA levels in the urine samples and monitor dynamic changes. The number of samples and the time points at which the urine samples are collected with respect to the treatment may vary depending on the required information.

In some embodiments, the method comprises collecting at least one urine sample from the subject prior to receiving the treatment and preparing the urine sample as described herein.

In accordance with some aspects, the present disclosure provides a method of preparing urine samples for characterizing cfDNA in at least one urine samples, the method comprising preparing at least one urine sample at a first time point and at least one urine sample at a second time point, wherein the subject received at least one dose of a treatment between the first time point and the second time point. In some embodiments, the methods comprise monitoring cfDNA levels in urine samples obtained from a subject by comparing cfDNA levels before and after initiation of a treatment, i.e. before and after administration of at least one dose of a treatment.

In accordance with some embodiments, the first time point is a time point prior to administering to a subject a first dose of a treatment and the second time point is a time point after administering to a subject a dose of the treatment. In other words, in such embodiments, the first time point is before initiation of a treatment and the second time point is a time point after administering to a subject a dose of the treatment, for example, the first dose, the second dose or any sequential administration thereof.

In some embodiments where the first time point is before initiation of a treatment, the method comprises collecting one or more urine samples. In some embodiments where the first time point is before initiation of a treatment, the method comprises collecting one, two, three, four, five, six urine samples at different multiple first time points. In some examples, each one of the different multiple first time points is at one day, two days, three days, four days, five days or six days or more prior to initiation of treatment.

In some embodiments, the method comprises preparing one or more urine samples at the second time point. In some embodiments, the second time point is after initiation of a treatment, the method comprises preparing one, two, three, four, five, six urine samples at different second time points. In some examples, each one of the different multiple first time points is at one day, two days, three days, four days, five days or six days, a week, 10 days, two weeks or more after initiation of treatment.

The time difference between the first time point or multiple first time points and the second time point or multiple second time points may be determined based upon various parameters, including the treatment duration, severeness of disease etc.

In accordance with some aspects, the present disclosure provides a method comprises contacting at least one urine sample collected from a subject at a first time point and at a second time point with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA and wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In accordance with some aspects, the present disclosure provides a method comprises collecting one or more urine samples from a subject at a first time point and at a second time point, contacting each one of the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA and wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In accordance with some aspects, the present disclosure provides a method comprises the steps of filtering at least one urine sample collected from a subject at a first time point and at a second time point to obtain a filtered urine sample, contacting the at least one filtered urine sample with the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA and wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In accordance with some aspects, the present disclosure provides a method comprises the steps of collecting one or more urine samples from a subject at a first time point and at a second time point, filtering the one or more urine samples to obtained a filtered urine sample, contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA and characterizing cfDNA and wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment. In some embodiments, the second time point is at least about one hour after the first time point, at times at least about 2 hours, at times at least about 3 hours, at times at least about 5 hours, at times at least about 7 hours, at times at least about 10 hours, at times at least about 12 hours, at times at least about 14 hours, at times at least about 15 hours, at times at least about 17 hours, at times at least about 20 hours, at times at least about 22 hours, at times at least about 24 hours, at times at least about 27 hours, at times at least about 30 hours, at times at least about 32 hours, at times at least about 35 hours, at times at least about 36 hours, at times at least about 38 hours, at times at least about 40 hours, at times at least about 42 hours, at times at least about 44 hours, at times at least about 45 hours, at times at least about 48 hours from the first time point.

In some embodiments, the second time point is between about one hour and about 80 hours after the first time point, at times between about one hour and about 72 hours, at times between about one hour and about 70 hours, at times between about one-hour to about 65 hours, at times between about one -hour to about 60 hours, at times between about one-hour to about 55 hours, at times between about one -hour to about 50 hours, at times between about one-hour to about 48 hours, at times between about one -hour to about 40 hours, at times between about one -hour hours to about 36 hours, at times between about one-hour to about 30 hours, at times between about one -hour to about 24 hours, at times between about one-hour to about 20 hours, at times between about one- hour to about 18 hours, at times between about one-hour to about 16 hours, at times between about one-hour to about 14 hours, at times between about one -hour to about 12 hours from the first time point.

In some embodiments, the second time point is between about ten hours and about 80 hours after the first time point, at times between about ten hours and about 72 hours, at times between about ten hours and about 70 hours, at times between about ten hours to about 65 hours, at times between about ten hours to about 60 hours, at times between about ten hours to about 55 hours, at times between about ten hours to about 50 hours, at times between about ten hours to about 48 hours, at times between about ten hours to about 40 hours, at times between about ten hours to about 36 hours, at times between about ten hours to about 30 hours, at times between about ten hours to about 24 hours from the first time point. In some embodiments, the second time point is about 12 hours from the first time point, at times about 18 hours from the first time point, at times about 24 hours from the first time point, at times about 30 hours from the first time point, at times about 36 hours from the first time point, at times about 40 hours from the first time point, at times about 48 hours from the first time point, at times about 50 hours from the first time point, at times about 55 hours from the first time point, at times about 60 hours from the first time point, at times about 65 hours from the first time point, at times about 70 hours from the first time point, at times about 72 hours from the first time point.

As appreciated and as noted above, any one of the first time point and/or the second time point may encompass one or more time points.

In accordance with some embodiments, the first time point and the second time point are both time points after initiation of a treatment. In other words, in such embodiments, the first time point is before administering at least one dose of a treatment (herein first dose) and the second time point is a time point after administering to a subject a dose of the treatment, for example, the first dose, the second dose or any sequential administration thereof.

Hence, in accordance with some aspects, the present disclosure provides a method for determining a personalized treatment regimen for a subject suffering from a pathological disorder by assessing responsiveness of the subject to a treatment regimen and/or monitoring disease progression of the subject.

In some embodiments, the method comprising comparing cfDNA levels at a first time point and at a second time point wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In some embodiments, the method comprising comparing cfDNA levels at a first time point and at a second time point wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment and classifying the subject. In some embodiments, classifying the subject is based on the dynamic changes in the level of cfDNA.

In some embodiments, classifying the subject is based on determining if the subject is responsive to the treatment (determine treatment efficacy).

In some embodiments, classifying the subject is based on determining if the subject is non-responsive to the treatment (determine treatment efficacy).

In some embodiments, the method comprises a step of administering the treatment to a subject classified as a responder.

In accordance with some further aspects, the present disclosure provides a method for determining a personalized treatment regimen for a subject suffering from a proliferative disorder, by assessing responsiveness of the subject to a treatment and/or monitoring disease progression of the subject. The methods comprising the following steps: (a) characterizing level of cfDNA in at least two urine samples prepared as described herein and (b) classifying the subject.

In accordance with some further aspects, the present disclosure provides a method for determining a personalized treatment regimen for a subject suffering from a proliferative disorder, by assessing responsiveness of the subject to a treatment and/or monitoring disease progression of the subject. The methods comprising the following steps: (a) characterizing level of cfDNA in at least two urine samples prepared as described herein (b) classifying the subject and (c) administering the treatment to a subject classified as a responder.

In accordance with some aspects, the present disclosure provides a method comprises contacting at least one urine sample collected from a subject at a first time point and at a second time point with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA, characterizing cfDNA and classifying the subject, wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment. In accordance with some aspects, the present disclosure provides a method comprises collecting one or more urine samples from a subject at a first time point and at a second time point, contacting each one of the at least one urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA, characterizing cfDNA and classifying the subject, wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In accordance with some aspects, the present disclosure provides a method comprises the steps of filtering at least one urine sample collected from a subject at a first time point and at a second time point to obtain a filtered urine sample, contacting the at least one filtered urine sample with the composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA, characterizing cfDNA and classifying the subject, wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In accordance with some aspects, the present disclosure provides a method comprises the steps of collecting one or more urine samples from a subject at a first time point and at a second time point, filtering the one or more urine samples to obtained a filtered urine sample, contacting each one of the at least one filtered urine sample with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, isolating a portion of the one or more urine sample, wherein the portion is suspected to comprise cfDNA, characterizing cfDNA and classifying the subject, wherein the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In some embodiments, classifying the subject by determining treatment efficacy (i.e. responder or non-responder) comprises identifying the treatment as being effective in the subject if the cfDNA level determined in the second time point is higher (increased) as compared to the level determined in the first time point. In some examples, the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In some examples, classifying the subject as a responder, i.e. identifying the treatment as being effective in the subject, if the cfDNA level determined in the second time point is higher (increased) as compared to the level determined in the first time point, the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In some examples, classifying the subject as a responder if the cfDNA level determined in the second time point is higher by at least 2-fold, at times by at least 3 -fold, at times by at least 4-folds, at times by at least 5-folds, at times by at least 7-folds, at times by at least 10-folds as compared to the level determined in the first time point, the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment.

In some examples, classifying the subject as a responder if the cfDNA level determined in the second time point is higher by at least 2-fold, at times by at least 3 -fold, at times by at least 4-folds, at times by at least 5-folds, at times by at least 7-folds, at times by at least 10-folds as compared to the level determined in the first time point, the first time point is before the subject received at least one dose of a treatment and the second time point is after the subject received at least one dose of a treatment and the second time is between 4hours to 72 hours after the first time point.

In some embodiments, an increase in the cfDNA level in the second time point as determined by the methods of the present disclosure may be by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,

36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,

51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,

66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,

81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 99.9%, 99.99%, 99.999%, 99.9999% as compared to the cfDNA level as determined in the first time point.

In some embodiments, an increase in the cfDNA level in the second time point as determined by the methods of the present disclosure may be by about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12- fold, about 15 -fold, about 17-fold, about 20-fold, about 22-fold, about 25 -fold, about 27- fold, about 30-fold, about 32-fold, about 35-fold, about 37-fold, about 40-fold, about 42- fold, about 45-fold, about 47-fold, about 50-fold, as compared to the cfDNA level as determined in the first time point.

In some embodiments, determining treatment efficacy comprises identifying the treatment as being effective (i.e. responsiveness to treatment) in the subject if the change in the cfDNA level determined in the second time point compared to the level determined in the first time point is above a predetermined change.

It should be noted that a predetermined change refers to a change that in some embodiments of the present disclosure, meets the requirements for providing both high sensitivity (true positive rate) and high specificity (true negative rate). Sensitivity relates to the rate of identification of the responder patients (samples) as such, out of a group of samples, whereas specificity relates to the rate of correct identification of responder samples as such, out of a group of samples. It should be noted that predetermined change may be also provided as control sample/s or alternatively and/or additionally, as standard curve/s that display predetermined standard values for responders, non-responders, and for subjects that display responsiveness to a certain extent (level of responsiveness, e.g., low, moderate and high).

For example, and as shown herein, the predetermined change reflects the result of a statistical analysis of cfDNA in pre-established populations of responder or nonresponder. Pre-established populations as used herein refer to population of patients known to be responsive to a treatment of interest, or alternatively, population of patients known to be non-responsive or drug-resistant to a treatment of interest.

It should be emphasized that the nature of the invention is such that the accumulation of further patient data may improve the accuracy of the presently provided threshold values, which are usually based on ROC (Receiver Operating Characteristic) curves generated according to the patient data using analytical software program.

In some embodiments, determining treatment efficacy comprises identifying the treatment as being effective in the subject if the ratio between the cfDNA level determined in the second time point and the level determined in the first time point is above a predetermined ratio. In some embodiments, the predetermined change and/or the predetermined ratio are determined in a control subject. The control subject in accordance with some embodiments, is a non-responder subject. Alternatively, the control subject in accordance with some embodiments, is a healthy volunteer not administered with treatment.

The control sample in accordance with some embodiments may be obtained by the use of spike-in with non-human DNA. In some embodiments, the spike-in control is scd- 2 from C. elegans

F2_spike_cDNAscd2 5' TTC TGC AAC GAA GCG CTT TG 3'

R2_spike_cDNAscd2 5' ACG GCA CTC CTT CAC AAA AG 3'

In accordance with some aspects, the present disclosure provides a method for assessing responsiveness of a subject to a treatment, monitoring effectiveness of treatment and disease progression. The method comprises the steps of (a) determining level of cfDNA in at least two temporally-separated urine samples prepared as described herein, and (b) calculating a rate of change of the level of cfDNA between at least two temporally-separated urine samples.

In some embodiments, the at least two temporally-separated urine samples comprises at least one urine sample obtained from the subject before the subject received at least one dose of the treatment and the temporally-separated urine sample is obtained from the subject after the subject received at least one dose of the treatment, the urine samples are prepared as described herein.

In some embodiments, responsiveness to the treatment is associated with a rate of change above a predetermined rate of change. In some embodiments, the predetermined rate of change for the level of cfDNA is calculated in two or more control samples.

In some embodiments, the predetermined rate of change for the level of cfDNA is calculated in a non-responder subject.

In some embodiments, the predetermined rate of change for the level of cfDNA is calculated in a responder subject.

In accordance with some other aspects, the present disclosure provides a method for assessing responsiveness of a subject to a treatment regimen, the method comprises the steps of:

-determining level of cfDNA in at least one urine sample prepared by the methods as described herein at a time point before the subject received at least one dose of the treatment,

-determining level of cfDNA in at least one urine sample prepared by the methods as described herein at a time point after the subject received at least one dose of the treatment, and

-determining whether the subject is responsiveness to treatment regimen.

In accordance with some further aspects, the present disclosure provides a method for assessing responsiveness of a subject to a treatment regimen, the method comprises the steps of:

-determining level of cfDNA in at least one urine sample prepared by the methods as described herein at a time point after the subject received at least one dose of the treatment, and calculating the rate of change of the level of cfDNA between the at least one sample obtained at a time point prior to the treatment and the at least one sample obtained at a time point after initiation of the treatment; wherein a rate of change below a predetermined rate of change indicates that the subject belongs to a pre-established non- responsive population and/or a rate of change above a predetermined rate of change indicates that the subject belongs to a pre-established non-responsive population.

In some embodiments, the method comprises comparing the rate of change to a control rate of change.

As used herein the term "comparing” denotes any examination of the parameter and/or values obtained in the samples of the invention as detailed throughout in order to determine the suitable treatment protocol. It should be noted that comparing according to the present invention encompasses the possibility to use a computer-based approach.

The term "response" or "responsiveness" to a certain treatment, specifically, treatment regimen, refers to an improvement in at least one relevant clinical parameter as compared to an untreated subject diagnosed with the same pathology (e.g., the same type, stage, degree and/or classification of the pathology), or as compared to the clinical parameters of the same subject prior to treatment with the indicated medicament.

The term “non responder” or "drug resistance" to treatment with a specific medicament, specifically, treatment regimen that comprise the disclosed modulators, refers to a patient not experiencing an improvement in at least one of the clinical parameter and is diagnosed with the same condition as an untreated subject diagnosed with the same pathology (e.g., the same type, stage, degree and/or classification of the pathology), or experiencing the clinical parameters of the same subject prior to treatment with the specific medicament.

In some examples, the subject is suffering from an infectious disease.

An infectious disease may be any one of protozoan diseases, viral diseases, bacterial diseases, parasitic diseases, fungal diseases and mycoplasma diseases. It should be appreciated that an infectious disease as used herein also encompasses any infectious disease caused by a pathogenic agent. Pathogenic agents include prokaryotic microorganisms, lower eukaryotic microorganisms, complex eukaryotic organisms, viruses, fungi, prions, parasites, yeasts, toxins and venoms. In some examples, the subject is suffering from an organ failure and underwent transplantation. The treatment in such case is the transplantation. In some examples, the subject is suffering from a kidney failure and underwent kidney transplantation.

The subject described herein is suffering or suspected to suffer from a pathological disorder.

The pathological disorder in accordance with the present disclosure encompasses a disorder associated with abnormal degradation or fragmentation of genetic material released into the bloodstream, typically originating from cells undergoing cell death or shedding, such as apoptotic or necrotic cells.

In some embodiments, the pathological disorder is a genetic (inherited) disorder.

In some other embodiments, the pathological disorder is an acquired disorder (not genetic disorder).

In some embodiments, the pathological disorder is proliferative disorder and associated pathologies.

In some embodiments, the subject is suspected to be or diagnosed with a proliferative disorder and/or any associated pathologies. In some embodiments, the subject is suffering from a proliferative disorder and/or any associated pathologies. The proliferative disorder is in accordance with some embodiments a malignant proliferative disorder.

As used herein, proliferative disorder is a disorder displaying hyper proliferation. This term means cell division and growth that is not part of normal cellular turnover, metabolism, growth, or propagation of the whole organism. Unwanted proliferation of cells is seen in tumors and other pathological proliferation of cells, does not serve normal function, and for the most part will continue unbridled at a growth rate exceeding that of cells of a normal tissue in the absence of outside intervention. A pathological state that ensues because of the unwanted proliferation of cells is referred herein as a "hyper proliferative disease" or "hyper proliferative disorder." It should be noted that the term “proliferative disorder”, “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. Malignancies of tissues or organs may produce solid tumors. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. In general, the diagnostic, prognostic and therapeutic methods of the present invention may be applicable for patients suffering of non-solid tumors as well as of solid tumors.

The proliferative disorder as used herein also encompasses metastatic property. In some embodiments, the subject is suspected to be or diagnosed with a metastatic proliferative disorder. In some embodiments, the subject is suffering from a metastatic proliferative disorder. The metastatic proliferative disorder refers to a metastatic cancer.As used herein, metastasis or metastatic disorder refers to spread of cancer cells from a primary (initial) site to a different or secondary site (often by way of the lymph system or bloodstream). Hence, a metastatic cancer, or metastatic tumor, is one that has spread from the primary site (where it started) into different area(s) of the body (secondary sites). Tumors formed from cells that have spread are called secondary tumors (metastases).

In some embodiments, the proliferative disorder may be any one of leukemias and lymphoma.

In some embodiments, the proliferative disorder may be any one of carcinoma, melanoma, sarcoma, glioma and blastoma.

In some embodiments, the proliferative disorder is a carcinoma. In some embodiments, the carcinoma is an adenocarcinoma, a basal cell carcinoma, or squamous cell carcinoma.

In some embodiments, the malignant proliferative disorder may be at least one primary and/or secondary malignancy of at least one of breast cancer, bladder cancer, kidney cancer, hepatocarcinoma cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, melanoma, sarcoma, and specifically, osteosarcoma, ovarian cancer, prostate cancer, thyroid cancer, cervical cancer, uterus cancer, laryngeal cancer, brain cancer, hematopoietic malignancies (non-solid lymphoma, leukemia, or multiple myeloma).

In some embodiments, the proliferative disorder is lung cancer (lung carcinoma). Lung cancer is one of the most common malignancies and a leading cause of cancer death.

In some embodiments, the lung carcinoma is a small-cell lung carcinoma (SCLC).

In some other embodiments, the lung carcinoma is non-small-cell lung carcinoma (NSCLC). The majority of lung cancer patients (-85%) are diagnosed with non-small-cell lung cancer (NSCLC).

In some embodiments, the subject is diagnosed with lung cancer. In some embodiments, the subject is diagnosed with a metastatic lung cancer.

In some embodiments, the subject is diagnosed with SCLC. In some embodiments, the subject is diagnosed with a metastatic SCLC.

In some embodiments, the subject is diagnosed with NSCLC. In some embodiments, the subject is diagnosed with a metastatic NSCLC. In some embodiments, the subject is diagnosed with NSCLC and metastasis in hemithorax, liver, bones.

As described herein, some of the drawbacks associated with cancer treatment results from resistance to treatment optionally due to mutations.

In some embodiments, the proliferative disorder comprises at least one mutation. In some other embodiments, the proliferative disorder comprising at least one mutation in a protein kinase. In some further embodiments, the proliferative is NSCLC comprising at least one mutation in a protein kinase.

In some embodiments, the disorder is a protein kinase-mediated disorder.

As appreciated, protein kinase selectively modifies other proteins by covalently adding a phosphate moiety to them (phosphorylation).

In some embodiments, the protein kinase is a serine/threonine kinase or a tyrosine kinase.

In some embodiments, the protein kinase is a tyrosine kinase.

A tyrosine protein kinase refers to an enzyme that can transfer a phosphate group from ATP to the tyrosine residues of specific proteins inside a cell and usually functions as an "on" or "off" switch in many cellular functions. In some embodiments, the tyrosine kinase protein is at least one of growth factor receptor (GFR), c-Met, c-Kit, PI3-kinase, PI3CA, epidermal growth factor receptor (EGFR), Platelet-derived growth factor receptor (PDGFR), insulin receptor and insulin-like growth factor 1 receptor (IGF1R), stem cell factor (SCF) receptor, Anaplastic lymphoma kinase (ALK), fibroblast growth factor receptor 1 (FGFR1), Proto-oncogene tyrosine-protein kinase (ROS1), RET, NTRF1, HER2 (ERBB2), Tropomyosin receptor kinase A (TrkA), MEK1, MEK2, MAP2K1, ABL, Discoidin domain receptor family, member 1 (DDR1), Discoidin domain receptor family, member 2 (DDR2), quinone reductase 2 (NQ02), Vascular endothelial growth factor receptor (VEGFR), c-RAF, b-RAF, Granulocyte colonystimulating factor (G-CSF), FLT3, Src, Epidermal growth factor receptor (EGFR), lymphocyte-specific protein tyrosine kinase (Lek), EPHA3, EPHA8, Mitogen-activated protein kinase 11 (MAPK11), zipper containing kinase AZK (ZAK), Fibroblast Growth Factor Receptor (FGFR), ROS1, C-MET, JAK1, JAK2, AXL, flt3, BTK, IDH2, or combinations thereof.

In some further embodiments, the proliferative disorder is NSCLC comprising at least one mutation in a protein kinase as compared to the wild type in the protein kinase.

In some further embodiments, the proliferative disorder is NSCLC comprising at least one mutation in at least one of GFR, EGFR, PDGFR, IGF1R, SCF, ALK or ROS as compared to the respective wild type protein kinase.

In some embodiments, the NSCLC is at least one of EGFR-mutated, ALK mutated or ROS 1 mutated NSCLC as compared to the respective wild type protein kinase.

In some embodiments, the proliferative disorder is brain cancer. In some embodiments, the proliferative disorder is metastatic brain cancer.

The present disclosure is not limited to a specific treatment and can be applicable to a variety of treatment protocols.

In some embodiments, the subject is suffering from a proliferative disorder and the treatment is an anticancer treatment (anti-proliferative therapy). As appropriated, the term anticancer treatment refers to any treatment intended for eliminating or killing cancer cells in the primary tumor, secondary tumor, or combination thereof as well as cells of any other proliferative disorder.

In some embodiments, the anticancer treatment may comprise at least one of chemotherapy, radiosurgery, radiation therapy, biological therapy, immune-therapy, hormone therapy, surgery, or any combination thereof. In some embodiments, the anticancer treatment is aimed at treating the primary, secondary tumors, or combination thereof and may be selected based on the methods described herein. In some embodiments, the anticancer treatment is a therapeutic agent being an anticancer drug (also denoted antineoplastic drug).

In some embodiments, the anticancer treatment is a therapeutic agent being an anticancer drug (also denoted antineoplastic drug).

The anticancer drug as used herein refers to an agent that is effective in treatment of malignant or cancerous disease.

In some embodiments, the anticancer drug is a cytotoxic anticancer drug.

In some embodiments, the anticancer drug is a chemotherapeutic drug.

In some embodiments, the anticancer drug is a non-specific anticancer drug.

A cytotoxic anticancer drug is considered as a drug that is unable to distinguish between cancer cells and naturally rapidly dividing normal cells in the body, and hence cause side effects such as bone marrow suppression, alopecia, and diarrhea.

In some embodiments, the non-specific anticancer drug is at least one of an alkylating agent, a platinum compound, an anti-metabolite compound, a mitotic spindle inhibitor, a topoisomerase inhibitor or a combination thereof.

In some embodiments, the anticancer drug is a drug that alters hormonal milieu. In some embodiments, the anticancer drug is tamoxifen.

In some embodiments, the anticancer drug is a targeted therapy. The term targeted therapy is a type of treatment that uses drugs or other substances to identify and attack specific types of cancer cells with less harm to normal cells. In some embodiments, the target drug is at least one of small molecule drugs, large molecule drug or combination thereof.

In some embodiments, the anticancer drug is a targeted anticancer drug.

In some embodiments, the anticancer drug is at least one of a large molecule or a small molecule.

In some embodiments, the anticancer drug is at least one large molecule drug.

In some embodiments, the large molecule is at least one of a monoclonal antibody, an immunotoxin or a combination thereof.

In some embodiments, the anticancer drug is a biological drug. A biological therapy uses substances made from living organisms to treat disease and may occur naturally in the body or may be made in the laboratory.

In some embodiments, the anticancer drug is at least one small molecule drug.

In some embodiments, the small molecule is at least one of a tyrosine kinase inhibitor (TKI), a proteosome inhibitor, a PARP inhibitor, a CdK inhibitor or any combination thereof.

In some embodiments, the treatment is or comprises at least one TKI.

In some embodiments, the anticancer drug is a TKI inhibitor.

In some embodiments, the at least one TKI is at least one of Imatinib, Gefitinib, Erlotinib, Dasatinib, Sunitinib, Adavosertib, Lapatinib, Osimertinib, Crizotinib, Alectininb, Ceritinib, Brigatinib, Lorlatinib, Ensartinib, Sorafenib, Nilotinib, Pazopanib, Ruxolitinib, Vemurafenib, Vandetanib, Regorafenib, Cabozantinib, Axitinib, Bosutinib, Ponatinib, Afatinib, Trametinib, Dabrafenib, Ibrutinib, Ceritinib, Lenvatinib, Osimertinib, Alectinib, Cobimetinib, Neratinib, Brigatinib, Acalabrutinib, Midostaurin, Tivozanib, Enasidenib, Encorafenib, Dacomitinib, Lorlatinib, Binimetinib, Larotrectinib, Gilteritinib, erdafitinib, Pexidartinib, Entrectinib, Zanubrutinib, Fedratinib, Tucatinib, Avapritinib, Ripretinib, Pemigatinib, Capmatinib, Selpercatinib, Pralsetinib, Selumetinib, Infigratinib, Tepotinib, Mobocertinib, Asciminib, Volasertib or any combination thereof. In some embodiments, the at least one TKI is at least one of Gefitinib, Erlotinib, Sorafenib, Crizotinib, Afatinib, Trametinib, Dabrafenib, Ceritinib, Ensartinib, Osimertinib, Alectinib, Brigatinib, Dacomitinib, Lorlatinib, Entrectinib, Capmatinib, Selpercatinib, Pralsetinib, Tepotinib, Mobocertinib or any combination thereof.

In some embodiments, the therapeutic methods of the disclosure are considered as first line treatment.

First-line treatment or first-line therapy refers to the initial, or first treatment recommended for a disease, including, inter alia, proliferative disorder. It is of note that first-line treatment is considered as a treatment that is expected to provide the best results with the fewest number of side effects for all patients.

In some embodiments, the therapeutic methods of the disclosure are for a subject who has been previously treated with a first-line treatment. In some embodiments, the therapeutic methods of the disclosure are considered as second line treatment.

In some embodiments, the subject to be treated by the methods of the disclosure has been previously treated with surgery, radiation therapy, targeted therapy, cytotoxic therapy or combination thereof.

In some embodiments, the subject has been treated with a TKI.

Second-line treatments are used when the first-line treatment failed to improve a cancer, or if it worked for a while and then the cancer progressed, and tend to be less effective.

In accordance with some other aspects, the present disclosure provides a method for treating lung cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a TKI.

In some embodiments, the subject was previously treated with a first line treatment.

It is to be understood that the terms "treat”, “treating”, “treatment" or forms thereof, as used herein, mean preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder. Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from a pathologic disorder. Specifically, providing a "preventive treatment" (to prevent) or a "prophylactic treatment" is acting in a protective manner, to defend against or prevent something, especially a condition or disease.

As indicated above, the methods and compositions provided by the present invention may be used for the treatment of a “pathological disorder”, specifically, proliferative disorders as specified by the invention and more specifically ling cancer. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein.

As indicated above, the composition may be used to stabilize cfDNA and hence reduce/prevent/inhibit cfDNA degradation such that there is essentially no degradation. When referring to essentially no degradation as used herein refers to the preservation of cfDNA in or obtained from a biological sample, such as a urine sample. Specifically, in the context of this disclosure, 'essentially no degradation' denotes the retention of cfDNA integrity with no occurrence of fragmentation, breakage, modification of cfDNA or any of the like. As appreciated, the degree of degradation may be quantified by measurable parameters, including but not limited to the percentage of intact cfDNA strands, base pair integrity, or any other suitable metric as determined by standard analytical techniques known in the art for example in comparison to a cfDNA from a biological sample without the composition described herein. The essentially no degradation may by an inhibition/reduction of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,

28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,

43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 99.9%, 99.99%, 99.999%, 99.9999% as compared to a stabilized cfDNA level.

The present invention relates to the treatment of subjects or patients, in need thereof. By “patient” or “subject in need” it is meant any organism who may be affected by the above-mentioned conditions, and to whom the therapeutic and prophylactic methods herein described are desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and rodents, specifically, murine subjects. More specifically, the methods of the invention are intended for mammals. By “mammalian subject” is meant any mammal for which the proposed therapy is desired, including human, livestock, equine, canine, and feline subjects, most specifically humans.

The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. In some embodiments, the term "about" refers to ± 10 %.

As used herein, the forms "a", "an" and "the" include singular as well as plural references unless the context clearly dictates otherwise.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments unless the embodiment is inoperative without those elements.

It should be noted that the various embodiments and examples detailed herein in connection with various aspects of the invention may be applicable to one or more aspects disclosed herein. It should be further noted that any embodiment described herein may be applied separately or in various combinations. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. The phrases “in another embodiment” or any refence made to embodiment as used herein do not necessarily refer to different embodiment, although it may. Thus, various embodiments of the invention can be combined (from the same or from different aspects) without departing from the scope of the invention. Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, kits and compositions disclosed herein as such methods steps, kits and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

NON-LIMITING EXAMPLES

Example 1: preparation of urine samples for determination of cfDNA

Urine samples (one or more) were collected and prepared using an exemplary kit shown in Fig. 1 as detailed herein below:

The kit was used as follows: urine sample was collected in the plastic cap (1) and 10 ml of the urine are pulled into the syringe (3), the 10 ml urine were filtered by a green syringe filter (4) into a tube containing small amount of brown-colored buffer with a blue cap comprising urine collection solution that is placed on the single tube stand (5). Following filtration, the tube was vortexed (6) until the solution becomes slightly brown. The tube was placed back to the single tube stand (5) for 5 min. After 5 min the tube was transferred to the magnet stand (7) for 2 min. While the tube was still on the magnet stand, the cap is gently opened and the urine was pulled out to the plastic cap using a plastic pipette (8) leaving small volume of liquid in the tube (labeled with a blue mark on the tube). The tube was labeled with the time, date, and code of patient and placed in the collecting stand (10) in the home fridge. The urine in (1) was discarded. At day 6 or 8, the six tubes were sent to the laboratory, where cfDNA was purified using Quick-DNA Urine Kit (Zymo Research) and quantified using real-time PCR. Primers for real-time PCR include F_TERT_3815 5' CCC TCC TTT GCC TTC CAC 3'; R_TERT_3875 5' GGT CAC TCC AAA TTC CCA GA 3'; F2_spike_cDNAscd2 5' TTC TGC AAC GAA GCG CTT TG 3'; R2_spike_cDNAscd2 5' ACG GCA CTC CTT CAC AAA AG 3'.

The urine collection solution comprises 300 pl buffer containing (5mM Tris pH7.8, O.lmM CaC12, 0.2mM ATP, 0.2mM DTT, 3.3pg/ml actin, 83pM latrunculin and 15 pl magnetic beads solution (Zymo Research D3061-2-1).

Example 2: cfDNA for assessing treatment responsiveness

Urine samples from five individuals were obtained using a kit described in Example 1 and the cfDNA was quantified.

Example 2A: EGFR positive NSCLC patient 1

Urine samples were collected from a patient diagnosed with EGFR positive NSCLC patient, three days prior to treatment initiation as baseline and four days on treatment with an EGFR inhibitor, Osimertinib as first-line treatment.

Fig. 2A shows a daily dynamic of total cfDNA levels in the urine samples. As can be seen, an increase in cfDNA as indicated by TERT copies/ml urine was observed one day after initiation of treatment.

Fig. 2B and 2C are PET-CT taken about one month before treatment (Fig. 2B) and 4 months after the treatment (Fig. 2C). As can be seen, the tumor’s shown in Fig. 2B by arrows do not appear in Fig. 2C. Hence, as can be seen from Fig. 2C, the patient responded to treatment.

Hence, the results suggest the observed increase in cfDNA of about 6 folds can be considered as indicative to responsiveness to treatment. Example 2B: EGFR positive NSCLC patient 2

Urine samples were collected from a patient diagnosed with EGFR positive NSCLC patient, one day prior to treatment initiation as baseline and four days after initiation of treatment with an EGFR inhibitor, Osimertinib as first-line treatment. Samples were not taken for week and followed by additional five samples from consecutive days.

Fig. 3A shows a daily dynamic of total cfDNA levels in the urine samples. As can be seen, an increase in cfDNA as indicated by TERT copies/ml urine was observed one day after initiation of treatment.

Fig. 3B and 3C are PET-CT about one month before treatment (Fig. 3B) and 2 months after treatment (Fig. 3C) with Osimertinib.

As can be seen from Fig. 3C, the tumor has disappeared suggesting that the patient responded to treatment.

Hence, the results suggest the observed increase in cfDNA of about 7 folds can be considered as indicative to responsiveness to treatment.

Example 2C: EGFR positive NSCLC patient 3

Urine samples were collected from a patient diagnosed with metastatic EGFR positive adenocarcinoma of the lung, two days prior to treatment with Osimertinib initiation as baseline and four days on treatment.

Fig. 4A shows a daily dynamic of total cfDNA levels in the urine samples. As can be seen, an increase in cfDNA as indicated by TERT copies/ml urine was observed one day after initiation of treatment. Radiology test indicated good response to treatment. Specifically, as can be see from Fig. 4B, a metastatic lytic bone lesion in vertebra T4 was noted on the pre-treatment CT studies and this lesion shows almost complete resolution with sclerotic changes on the follow-up CT (Fig. 4C).

Hence, the results suggest the observed increase in cfDNA of about 16 folds can be considered as indicative to responsiveness to treatment. Example 2D: EML4-ALK positive NSCLC patient 4

Urine samples were collected from a patient diagnosed with brain metastasis, prior to brain radiation as baseline and six days after radiation.

Fig. 5 shows a daily dynamic of total cfDNA levels in the urine samples. The two urine samples collected before the irradiation showed variations in the cfDNA as indicated by TERT copies/ml and this variation was considered as a base line. As can be seen, a significant increase of at least about 4-8 folds in cfDNA was observed abut 36 hours after radiation treatment.

Hence, the results suggest the observed increase in cfDNA can be considered as indicative to responsiveness to treatment.

Example 3: Control healthy volunteer

Urine samples were collected a healthy individual.

Fig. 6 shows a daily dynamic of total cfDNA levels in the urine samples. As can be seen, no change in the cfDNA level was observed with time.

Claims

CLAIMS:

1. A composition comprising one or more means to bind cfDNA and one or more cfDNA stabilization reagents.

2. The composition of claim 1 for use in stabilizing cfDNA in a urine sample or fractions thereof.

3. The composition of claim 1 or 2, wherein the one or more means to bind cfDNA is or comprises a solid support.

4. The composition of claim 1 or 2, wherein said one or more means to bind cfDNA is or comprises magnetic beads, super magnetic beads, magnetic nanoparticles or a combination thereof.

5. The composition of claim 4, wherein said one or more means to bind cfDNA is or comprises magnetic beads.

6. The composition of claim 1 or 2, wherein the one or more cfDNA stabilization reagents is or comprises at least one DNase inhibitor.

7. The composition of claim 6, wherein the at least one DNase inhibitor is or comprises actin filament.

8. The composition of claim 1 or 2, wherein the one or more cfDNA stabilization reagents is or comprises an actin filament inhibitor.

9. The composition of claim 8, wherein the actin filament inhibitor is at least one of substoichiometric cytochalasin B (CB), cytochalasin D, latrunculin or a combination thereof.

10. The composition of claim 9, wherein the actin filament inhibitor is latrunculin.

11. The composition of claim 1 having a pH of above about 7.

12. The composition of claim 11, having a pH of between about 7 and about 8.

13. The composition of any one of claims 1 to 12, comprising CaCh.

14. The composition of any one of claims 1 to 12, comprising ATP.

15. The composition of any one of claims 1 to 12, comprising DTT.

16. The composition of any one of claims 2 to 15 for use in characterizing cfDNA in the urine sample or a fraction thereof.

17. A composition comprising magnetic beads, actin, latrunculin, CaCh, ATP and DTT.

18. The composition of claim 17 for use in (i) stabilizing total cfDNA in a urine sample, (ii) characterizing cfDNA in the urine sample or a fraction thereof or (iii) a combination thereof.

19. A kit comprising a one or more container means, at least one of said one or more container means comprises a composition comprising one or more means to bind cfDNA and/or one or more cfDNA stabilization reagents, and optionally comprising instructions for preparing one or more urine samples for cfDNA characterization.

20. The kit of claim 19, wherein at least one of said one or more container means comprises a composition comprising one or more means to bind cfDNA and one or more cfDNA stabilization reagents.

21. The kit of any one of claim 19 or 20, wherein said composition is as defined in any one of claims 1 to 18.

22. The kit of any one of claims 19 to 21 comprising one or more filters.

23. The kit of any one of claims 19 to 21 comprising one or more urine collection means suitable for holding urine samples.

24. The kit of claim 23, wherein said instructions comprising collecting one or more urine samples in said urine collection means.

25. The kit of claim 24, wherein said instructions comprising filtering one or more urine samples to obtain one or more filtered urine samples.

26. The kit of claim 24 or 25, wherein said instructions comprising contacting said urine sample or filtered urine samples with the composition.

27. The kit of any one of claims 19 to 26, wherein said instructions comprises collecting at least one urine sample from a subject at a first time point and collecting at least one urine sample from said subject at a second time point, wherein said subject was administrated with at least one dose of a treatment between the first time point and the second time point.

28. The kit of any one of claims 19 to 27, wherein said instructions comprises isolating a fraction of said one or more urine samples or said one or more filtered urine samples, wherein said fraction is suspected to comprise total cfDNA.

29. A method comprising contacting one or more urine samples with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents, the one or more urine samples have been collected from a subject.

30. A method comprising collecting one or more urine samples from a subject and contacting each one of the a one or more urine samples with a composition comprising at least one means of binding cfDNA and one or more cfDNA stabilization reagents.

31. The method of claim 29 or 30 wherein said composition is defined in any one of claims 1 to 18.

32. The method of any one of claims 29 to 31 , comprising collecting said one or more urine samples are collected after the first urine of the day.

33. The method of any one of claims 29 to 32, comprising filtering said one or more urine samples prior to said contacting.

34. The method of any one of claims 29 to 33, comprising isolating a portion of the urine sample or the filtered urine sample, wherein said portion is suspected to comprise cfDNA.

35. The method of any one of claims 29 to 34, comprising characterizing cfDNA in said portion.

36. The method of claim 35, wherein said characterizing comprising contacting said portion with at least one cfDNA specific detecting molecules.

37. The method of any one of claims 29 to 36, comprising collecting at least one urine sample from said subject at a first time point and at least one other urine sample from said subject at a second time point, wherein said subject received at least one dose of a treatment between said first time point and said second time point.

38. The method of claim 37, wherein said first time point is before said subject received at least one dose of a treatment.

39. The method of claim 37, wherein said second time point is after said subject received at least one dose of a treatment.

40. The method of any one of claims 29 to 39 for assessing responsiveness of said subject to a treatment regimen by determining whether said subject is responsiveness to treatment regimen.

41. The method of claim 40 comprising calculating the rate of change of said level of cfDNA between the at least one sample obtained at a time point prior to said treatment and the at least one sample obtained at a time point after initiation of said treatment; wherein a rate of change above a predetermined rate of change indicates that said subject belongs to a responsive population.

42. The method of claim 41, wherein said predetermined rate of change is at least a three-fold increase.

43. The method of claim 42, wherein the subject is classified as a responder if at least a three -fold increase in the TERT per ml urine is observed between the first time point and the at second time point, such that the subject received at least one dose of a treatment between the first time point and the second time point.

44. The method of any one of claims 29 to 43, wherein said subject is suffering from a pathological disorder.

45. The method of claim 44, wherein the pathological disorder is a proliferative disorder or a metastatic proliferative disorder.

46. The method of claim 45, wherein said proliferative disorder is a carcinoma.

47. The method of claim 46, wherein said carcinoma is an adenocarcinoma, a basal cell carcinoma, or squamous cell carcinoma.

48. The method of claim 47, wherein said proliferative disorder is at least one primary and/or secondary malignancy of at least one of breast cancer, bladder cancer, kidney cancer, hepatocarcinoma cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, melanoma, sarcoma, and specifically, osteosarcoma, ovarian cancer, prostate cancer, thyroid cancer, cervical cancer, uterus cancer, laryngeal cancer, brain cancer, hematopoietic malignancies (non-solid lymphoma, leukemia, or multiple myeloma).

49. The method of claim 48, wherein the proliferative disorder is lung cancer (lung carcinoma).

50. The method of claim 49, wherein said lung cancer is a non-small-cell lung cancer (NSCLC).

51. The method of any one of claims 45 to 50, wherein said proliferative disorder is characterized by comprising at least one mutation in at least one protein kinase.

52. The method of claim 51 , wherein the protein kinase is a tyrosine kinase.

53. The method of claim 51 or 52, wherein the at least one mutation in the protein kinase is at least one of growth factor receptor (GFR), c-Met, c-Kit, PI3-kinase, PI3CA, epidermal growth factor receptor (EGFR), Platelet-derived growth factor receptor (PDGFR), insulin receptor and insulin-like growth factor 1 receptor (IGF1R), stem cell factor (SCF) receptor, Anaplastic lymphoma kinase (ALK), fibroblast growth factor receptor 1 (FGFR1), Proto-oncogene tyrosine-protein kinase (ROS1), RET, NTRF1, HER2 (ERBB2), Tropomyosin receptor kinase A (TrkA), MEK1, MEK2, MAP2K1, ABL, Discoidin domain receptor family, member 1 (DDR1), Discoidin domain receptor family, member 2 (DDR2), quinone reductase 2 (NQO2), Vascular endothelial growth factor receptor (VEGFR), c-RAF, b-RAF, Granulocyte colony-stimulating factor (G- CSF), FLT3, Src, Epidermal growth factor receptor (EGFR), lymphocyte-specific protein tyrosine kinase (Lek), EPHA3, EPHA8, Mitogen-activated protein kinase 11 (MAPK11), zipper containing kinase AZK (ZAK), Fibroblast Growth Factor Receptor (FGFR), ROS1, C-MET, JAK1, JAK2, AXL, flt3, BTK, IDH2, or combinations thereof.

54. The method of any one of claims 37 to 53, wherein said treatment is or comprises at least one tyrosine kinase inhibitor (TKI).

55. The method of claim 54, wherein said at least one TKI is at least one of Imatinib, Gefitinib, Erlotinib, Dasatinib, Sunitinib, Adavosertib, Lapatinib, Osimertinib, Crizotinib, Alectininb, Ceritinib, Brigatinib, Lorlatinib, Ensartinib, Sorafenib, Nilotinib, Pazopanib, Ruxolitinib, Vemurafenib, Vandetanib, Regorafenib, Cabozantinib, Axitinib, Bosutinib, Ponatinib, Afatinib, Trametinib, Dabrafenib, Ibrutinib, Ceritinib, Lenvatinib, Osimertinib, Alectinib, Cobimetinib, Neratinib, Brigatinib, Acalabrutinib, Midostaurin, Tivozanib, Enasidenib, Encorafenib, Dacomitinib, Lorlatinib, Binimetinib, Larotrectinib, Gilteritinib, erdafitinib, Pexidartinib, Entrectinib, Zanubrutinib, Fedratinib, Tucatinib, Avapritinib, Ripretinib, Pemigatinib, Capmatinib, Selpercatinib, Pralsetinib, Selumetinib, Infigratinib, Tepotinib, Mobocertinib, Asciminib or any combination thereof.

56. The method of claim 55, wherein said at least one TKI is at least one of Gefitinib, Erlotinib, Sorafenib, Crizotinib, Afatinib, Trametinib, Dabrafenib, Ceritinib, Ensartinib, Osimertinib, Alectinib, Brigatinib, Dacomitinib, Lorlatinib, Entrectinib, Capmatinib, Selpercatinib, Pralsetinib, Tepotinib, Mobocertinib or any combination thereof.