Prognostic and treatment response predictive method

Abstract

The present disclosure provides methods of predicting whether a subject is at risk of developing resistance to a treatment for chronic myelogenous leukaemia (CML) and / or progressing to blast crisis phase, wherein the subject has CML or has been diagnosed with CML. The present disclosure also provides methods of selecting a subject for treatment with a given therapy, wherein the subject has chronic myelogenous leukaemia (CML) or has been diagnosed with CML. The present disclosure also provides kits useful for performing said methods.

Description

[0001] PROGNOSTIC AND TREATMENT RESPONSE PREDICTIVE METHOD

[0002] This application claims priority from SG 10202403000P filed 26 September 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present disclosure relates to the field of cancer therapy, more specifically, methods of diagnosis and prognosis and methods of medical treatment and prophylaxis.

[0005] Background

[0006] Blast crisis (BC) chronic myeloid leukemia (CML) remains a major challenge in the management of CML patients as survival is usually measured in months. The ability to confidently identify chronic phase (CP) patients at high-risk of BC transformation may be life-saving, since it would prompt closer clinical monitoring and more aggressive treatment during CP, including stem cell transplantation, when such modalities are more effective (1 ,2). However, high-confidence predictions for future BC transformation are not currently possible, and will require the elucidation of pre-treatment factors that determine disease progression.

[0007] Therefore there remains a need for methods to identify chronic phase CML patients at risk of disease progression in order to inform appropriate treatment options.

[0008] Summary

[0009] In a first aspect, the invention relates to a method for predicting whether a subject is at risk of developing resistance to a treatment for chronic myelogenous leukaemia (CML) and / or progressing to blast crisis phase, wherein the subject has CML or has been diagnosed with CML, the method comprising: determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject; wherein (i) an increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML, (ii) an increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML, (iii) the presence of inflamed HSPCs, and / or (iv) the presence of inflamed lymphoid progenitor cells, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0010] Also disclosed is a method for predicting whether a subject is at risk of developing resistance to a treatment for chronic myelogenous leukaemia (CML) and / or progressing to blast crisis phase, wherein the subject has CML or has been diagnosed with CML, the method comprising: determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject; wherein (i) an increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML, (ii) an increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML, (iii) the presence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and / or (iv) the presence of inflamed lymphoid progenitor cells, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0011] In some embodiments, the method further comprises: determining (v) the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject, wherein a reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion in chronic phase CML patients who subsequently respond to treatment for CML, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0012] In some embodiments, the sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:

[0013] (i) a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3%,

[0014] (ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs equal to or greater than 5%,

[0015] (iii) an increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently respond to treatment for CML,

[0016] (iv) an increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0017] (v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 51 %.

[0018] In some embodiments, the sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:

[0019] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently progress to blast crisis phase; (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;

[0020] (iii) a similar or increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,

[0021] (iv) a similar or increased number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0022] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0023] In some embodiments, the sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:

[0024] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently progress to blast crisis phase;

[0025] (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;

[0026] (iii) a similar or increased number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive relative to the average number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,

[0027] (iv) a similar or increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0028] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0029] In some embodiments, the treatment for CML is a tyrosine kinase inhibitor (TKI). In some embodiments, the treatment for CML comprises a tyrosine kinase inhibitor (TKI).

[0030] In a further aspect, the invention relates to a method of selecting a subject for treatment, wherein the subject has chronic myelogenous leukaemia (CML) or has been diagnosed with CML, the method comprising:

[0031] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (Hi) the presence or absence of inflamed HSPCs, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;

[0032] (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) is increased relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is increased relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML, (iii) inflamed HSPCs are present, and (iv) inflamed lymphoid progenitor cells are present; wherein the treatment comprises one or more of a second-generation TKI, a third-generation TKI, a STAMP inhibitor, a combination of a TKI and chemotherapy, a combination of a TKI and one or more steroids, or a stem cell transplant.

[0033] Also disclosed herein is a method of selecting a subject for treatment, wherein the subject has chronic myelogenous leukaemia (CML) or has been diagnosed with CML, the method comprising:

[0034] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;

[0035] (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells is increased relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is increased relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML, (iii) inflamed HSPCs and / or inflamed megakaryocytic progenitors are present, and (iv) inflamed lymphoid progenitor cells are present; wherein the treatment comprises one or more of a second-generation TKI, a third-generation TKI, a STAMP inhibitor, a combination of a TKI and chemotherapy, a combination of a TKI and one or more steroids, or a stem cell transplant.

[0036] In some embodiments, the method further comprises (c) administering the treatment to a subject selected for treatment in step (b).

[0037] In some embodiments, step (a) further comprises determining the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject; and wherein step (b) further comprises selecting the subject for treatment if the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells is reduced relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML.

[0038] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0039] (i) a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3%,

[0040] (ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs equal to or greater than 5%,

[0041] (iii) an increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently respond to treatment for CML,

[0042] (iv) an increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0043] (v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 51 %.

[0044] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0045] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently progress to blast crisis phase;

[0046] (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;

[0047] (iii) a similar or increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,

[0048] (iv) a similar or increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0049] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0050] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0051] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently progress to blast crisis phase; (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;

[0052] (iii) a similar or increased number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive relative to the average number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,

[0053] (iv) a similar or increased number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0054] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0055] In a further aspect, the invention relates to a method of selecting a subject for treatment, wherein the subject has chronic myelogenous leukaemia (CML) or has been diagnosed with CML, the method comprising:

[0056] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;

[0057] (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) is reduced relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently progress to blast crisis phase, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is reduced relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase, (iii) inflamed HSPCs are not present, and (iv) inflamed lymphoid progenitor cells are not present; wherein the treatment comprises one or more of a first-generation TKI or a second-generation TKI.

[0058] Also disclosed herein is a method of selecting a subject for treatment, wherein the subject has chronic myelogenous leukaemia (CML) or has been diagnosed with CML, the method comprising:

[0059] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;

[0060] (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells is reduced relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently progress to blast crisis phase, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is reduced relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase, (iii) inflamed HSPCs and / or inflamed megakaryocytic progenitors are not present, and (iv) inflamed lymphoid progenitor cells are not present; wherein the treatment comprises one or more of a first-generation TKI or a second-generation TKI.

[0061] In some embodiments, the method further comprises (c) administering the treatment to a subject selected for treatment in step (b).

[0062] In some embodiments, step (a) further comprises determining the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject; and wherein step (b) further comprises selecting the subject for treatment if the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells is increased relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0063] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0064] (i) a proportion of megakaryocytic progenitor cells among HSPCs less than or equal to 3%,

[0065] (ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs less than or equal to than 5%,

[0066] (iii) a reduced number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently progress to blast crisis phase,

[0067] (iv) a reduced number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0068] (v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells equal to or greater than 51 %.

[0069] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0070] (i) a similar or reduced number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML;

[0071] (ii) a similar or reduced number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML; (iii) a similar or reduced number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently respond to treatment for CML,

[0072] (iv) a similar or reduced number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0073] (v) a similar or increased number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML.

[0074] In some embodiments, the sample obtained from a subject selected for treatment comprises one or more of:

[0075] (i) a similar or reduced number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently respond to treatment for CML;

[0076] (ii) a similar or reduced number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML;

[0077] (iii) a similar or reduced number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive relative to the average number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently respond to treatment for CML,

[0078] (iv) a similar or reduced number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0079] (v) a similar or increased number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML.

[0080] In some embodiments,

[0081] (i) determining the number / proportion of megakaryocytic progenitor cells comprises determining the number / proportion of CD42A+ cells among HSPCs; and / or

[0082] (ii) determining the number / proportion of lymphoid progenitor cells comprises determining the number / proportion of CD19+ and / or CD10+ cells among CD34+ HSPCs; and / or

[0083] (v) determining the number / proportion of adaptive NK cells comprises determining the number / proportion of NKG2C+ cells among CD56dimCD16bri9htNK cells.

[0084] In some embodiments,

[0085] (i) determining the number / proportion of megakaryocytic progenitor cells comprises determining the number / proportion of CD42A+ cells among HSPCs and / or CD45+ hematopoietic cells; and / or (ii) determining the number / proportion of lymphoid progenitor cells comprises determining the number / proportion of CD19+ and / or CD10+ cells among CD34+ HSPCs; and / or

[0086] (v) determining the number / proportion of adaptive NK cells comprises determining the number / proportion of NKG2C+ cells among CD56dimCD16bri9htNK cells.

[0087] In some embodiments,

[0088] (i) determining the number / proportion of megakaryocytic progenitor cells comprises determining the number / proportion of CD42A+ cells among lin- CD45+ CD34+ cells; and / or

[0089] (ii) determining the number / proportion of lymphoid progenitor cells comprises determining the number / proportion of CD19+ CD10+ cells among lin- CD45+ CD34+ CD38+ cells; and / or

[0090] (iii) determining the number / proportion of lin- CD45+ CD34+ cells that are pSTATI -positive; and / or

[0091] (iv) determining the number / proportion of lin- CD45+ CD34+ CD42A+ cells that are IFITM1- positive; and / or

[0092] (v) determining the number / proportion of adaptive NK cells comprises determining the number / proportion of NKG2C+ cells among lin- CD45+ CD56dimCD16bri9htNK cells.

[0093] In some embodiments,

[0094] (i) determining the number / proportion of megakaryocytic progenitor cells comprises determining the number / proportion of CD42A+ cells among CD45+ lin- CD34+ cells; and / or

[0095] (ii) determining the number / proportion of lymphoid progenitor cells comprises determining the number / proportion of CD19+ CD10+ cells among CD45+ lin- CD34+ CD38+ cells; and / or

[0096] (iii) determining the number / proportion of inflamed HSPCs comprises determining the number / proportion of CD45+ lin- CD34+ cells that are pSTATI -positive; and / or

[0097] (iv) determining the number / proportion of inflamed megakaryocytic progenitors comprises determining number / proportion of CD45+ lin- CD34+ CD42A+ cells that are pSTATI -positive;

[0098] (v) determining the number / proportion of inflamed lymphoid progenitor cells comprises determining number / proportion of CD45+ lin- CD34+ cells that are IFITMI-positive; and / or

[0099] (v) determining the number / proportion of inflamed lymphoid progenitor cells comprises determining number / proportion of CD45+ lin- CD34+ CD19+ CD10+ cells that are IFITMI-positive

[0100] (vii) determining the number / proportion of adaptive NK cells comprises determining the number / proportion of NKG2C+ cells among CD45+ CD3- CD14- CD19- CD56dimCD16bri9htNK cells.

[0101] In some embodiments, the patient sample has been obtained from peripheral blood or bone marrow.

[0102] In a further aspect, the invention relates to a kit for performing a method disclosed herein. In some embodiments, the kit comprises a plurality of antigen-binding molecules, wherein the plurality of antigenbinding molecules is capable of detecting one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38, CD45RA, CD42A, CD10, CD90, CD123, pSTATI and IFITM1 , and optionally one or more of CD71 , CD105, CD253A, CD56, NKG2A and / or NKG2C. Description

[0103] The present invention is based on the unexpected discovery that resistance to treatment (such as tyrosine kinase inhibitor (TKI) treatment) and the risk of blast crisis progression can be predicted in CML patients prior to the start of a treatment (e.g. at the time of CML diagnosis or subsequent to a CML diagnosis). Current clinical assessments are based on clinical parameters, such as age, spleen size and blood cell counts during treatment. The present invention is based on the use of biomarkers, which provides an accurate predictive test of treatment resistance that can be carried out at the time of CML diagnosis or subsequent to a CML diagnosis, allowing timely interventions that are more effective for high-risk patients.

[0104] Chronic Myelogenous Leukaemia (CML)

[0105] The present disclosure relates to methods for predicting whether a subject is at risk of developing resistance to a treatment for chronic myelogenous leukaemia (CML) and / or progressing to blast crisis phase.

[0106] Chronic myelogenous leukaemia (CML) is also known as chronic myeloid leukaemia, chronic granulocytic leukaemia, or Philadelphia (Ph*) / BCR-ABL1 -positive chronic myeloid leukaemia. CML is characterised by the chromosomal translocation t(9;22) (q34.1 ;q11 .2), resulting in the BCR-ABL1 fusion gene and formation of the Philadelphia chromosome (Ph*). CML is also characterised by the unregulated proliferation of blast cells in the bone marrow and the accumulation of blast cells in the blood. Typically, CML begins in a chronic phase, and then progresses to an accelerated phase, followed by a blast crisis phase.

[0107] In the chronic phase, CML is most stable and develops slowly. Patients with chronic phase CML have predominantly fully functioning blood cells with a low level of blast cells (less than 10% of the cells in the blood and bone marrow). Patients with chronic phase CML may also have a lower level of red blood cells (anaemia) and a higher level of platelets than is normal, but patients may have few or no symptoms.

[0108] As used herein, a “blast cell” refers to an early hematopoietic cell from the lymphoid (lymphocytes) or myeloid (erythrocytes, thrombocytes, monocytes, neutrophils, basophils, eosinophils) cell lines. Blast cells are produced by stem cells in the bone marrow. A blast cell may be a lymphoid blast cell (e.g. lymphoblast) or a myeloid blast cell (e.g., myeloblast). In healthy individuals, the bone marrow contains no more than 5% blast cells, and the peripheral blood contains no blast cells.

[0109] Accordingly, as used herein, “chronic phase CML” refers to CML where less than 10% of the cells in the blood and bone marrow are blast cells. In some embodiments, the chronic phase CML is asymptomatic or associated with mild symptoms. A chronic phase CML patient may be identified according to one or more of these criteria. For example, a chronic phase CML patient may be a patient where less than 10% of the cells in the blood and bone marrow are blast cells, or where the CML is asymptomatic or associated with mild symptoms. According to the MD Anderson Cancer Center (MDACC) criteria, accelerated phase, may be defined by the following criteria: peripheral blood myeloblasts of >15% and <30%; peripheral blood myeloblasts and promyelocytes combined of >30%; peripheral blood basophils of >20%; a platelet count of <100 x 109 / L unrelated to therapy; or additional clonal cytogenetic abnormalities in Ph+ cells. Accelerated phase may be accompanied by symptoms such as weight loss, fatigue, and an enlarged spleen (also known as splenomegaly). In addition, treatment may be less effective during the accelerated phase, as characterised by persistent or increasing abnormal blood cell counts despite treatment (e.g., leukocytosis (>10x109 / L), thrombocytosis (>1000x109 / L), or thrombocytopenia (<100x109 / L) unrelated to therapy, 20% or more basophils, or 10%-19% blast cells). The accelerated phase may also be accompanied by additional chromosomal abnormalities (in addition to the Philadelphia chromosome) such as second Ph* chromosome, trisomy 8, isochromosome 17q, trisomy 19, complex karyotype, or 3q26.2 abnormalities.

[0110] According to the International Bone Marrow Transplant Registry (IBMTR) criteria, blast crisis phase (also known as the blastic phase, the acute phase, the blast phase, blast crisis, or blast transformation), may be defined as having 30% or more blast cells within the cells in the blood and / or bone marrow, or by the presence of extramedullary infiltrates of leukemic cells. The blast crisis phase is associated with severe symptoms (such as increasing or persistent splenomegaly), increased resistance to treatment, and a poor prognosis. The blast crisis phase may be accompanied by additional chromosomal abnormalities (in addition to the Philadelphia chromosome) such as second Ph* chromosome, trisomy 8, isochromosome 17q, trisomy 19, complex karyotype, or 3q26.2 abnormalities. The blast crisis phase can occur as a myeloid blast crisis (where the blast cells are myeloblasts) or a lymphoid blast crisis (where the blast cells are lymphoblasts). In some cases, the blast cells in the blast crisis phase are a mixture of myeloblasts and lymphoblasts.

[0111] Accordingly, as used herein, “blast crisis phase CML” (also known as “blastic phase CML”, “acute phase CML, “blast phase CML” or “blast transformation CML”) refers to CML where 30% or more of the cells in the blood and / or bone marrow are blast cells, or where extramedullary infiltrates of leukemic cells are present. In some embodiments, blast crisis phase CML is as defined by appropriate diagnostic criteria (e.g. by the International Bone Marrow Transplant Registry (IBMTR) criteria (see for example, Gambacorti-Passerini & le Coutre, Chronic Myelogenous Leukemia in De Vita, Hellman and Rosenberg’s Cancer: Principles & Practice of Oncology (12thEdition) 2022:1773-1784, which is hereby incorporated by reference in its entirety). In some embodiments, extramedullary infiltrates of leukemic cells are present in the lymph node, soft tissue and / or CNS. In some embodiments, the blast crisis phase CML is myeloid blast crisis phase CML. In some embodiments, the blast crisis phase CML is lymphoid blast crisis phase CML. In some embodiments, the blast crisis phase CML is characterised by a mixture of myeloblasts and lymphoblasts.

[0112] A blast phase CML patient (or a chronic phase CML patient that has progressed to the blast crisis phase) may be a patient where 30% or more of the cells in the blood and / or bone marrow are blast cells, or a patient where extramedullary infiltrates of leukemic cells are present. In some embodiments, a blast phase CML patient (or a chronic phase CML patient that has progressed to the blast crisis phase) may be a patient according to an appropriate diagnostic criteria of blast phase CML (e.g. by the International Bone Marrow Transplant Registry (IBMTR) criteria (see for example, Gambacorti-Passerini & le Coutre, Chronic Myelogenous Leukemia in De Vita, Hellman and Rosenberg’s Cancer: Principles & Practice of Oncology (12thEdition) 2022:1773-1784, which is hereby incorporated by reference in its entirety). A blast crisis phase CML patient may have CML that is characterised by a myeloid blast crisis. A blast crisis phase CML patient may have CML characterised by a lymphoid blast crisis. A blast crisis phase CML patient (or a chronic phase CML patient that has progressed to the blast crisis phase) may have increasing or persistent splenomegaly, increased resistance to treatment (such as TKI treatment), and / or a poor prognosis. A blast crisis phase CML patient (or a chronic phase CML patient that has progressed to the blast crisis phase) may be unresponsive to treatment (e.g. TKI treatment, STAMP inhibitor treatment). A blast crisis phase CML patient (or a chronic phase CML patient that has progressed to the blast crisis phase) may have additional chromosomal abnormalities (in addition to the Philadelphia chromosome) such as second Ph* chromosome, trisomy 8, isochromosome 17q, trisomy 19, complex karyotype, or 3q26.2 abnormalities.

[0113] Diagnostic and prognostic methods, and patient selection

[0114] The present disclosure also provides diagnostic, prognostic and predictive methods in connection with the cancers described herein (e.g. CML).

[0115] The methods may be performed in vitro on a sample obtained from a subject, or following processing of a sample obtained from a subject. Once the sample is collected, the subject is not required to be present for the in vitro method to be performed, and therefore the method may be one which is not practised on the human or animal body.

[0116] A sample may be taken from any tissue or bodily fluid. A sample may comprise or may be derived from: a quantity of blood; a quantity of serum derived from a subject’s blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a tissue sample or biopsy (e.g. a bone marrow tissue sample or biopsy); pleural fluid; cerebrospinal fluid (CSF); or cells isolated from a subject. In some embodiments, the sample may be obtained or derived from a tissue or tissues which are affected by the disease / condition (e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease / condition). In some embodiments, the sample may be obtained or derived from a cancer, tumor, or cells thereof. In some embodiments, in a method according to the present disclosure the sample comprises or is derived from a quantity of blood (e.g. peripheral blood). In some embodiments, in a method according to the present disclosure the sample comprises or is derived from bone marrow (e.g. a bone marrow tissue sample or biopsy). In some embodiments, in a method according to the present disclosure the sample comprises mononuclear cells. For example, a sample (e.g. a blood or bone marrow sample) may be processed to separate / purify the mononuclear cells. In some embodiments, in a method according to the present disclosure the sample comprises bone marrow mononuclear cells (BM-MNCs) or peripheral blood mononuclear cells (PB- MNCs).

[0117] The methods may be performed for the purpose of diagnosing a cancer (e.g. a cancer described herein). The methods may be performed for the purpose of prognosing / predicting the likely progression of a cancer (e.g. CML). The methods may be performed for the purpose of prognosing / predicting the likely response of a subject to therapeutic / prophylactic intervention as described herein. The methods may be useful to predict the likely response to a given therapeutic / prophylactic intervention, e.g. in terms of efficacy and / or resistance, and may therefore by useful for supporting clinical decision-making. The methods may be performed for the purpose of identifying / selecting a subject for therapeutic / prophylactic intervention as described herein. The methods may be performed for the purpose of assessing / determining / monitoring a subject’s response to a therapeutic / prophylactic intervention as described herein.

[0118] In some embodiments, the methods comprise evaluating a cancer (e.g. CML) to determine whether it comprises hallmark features indicative of poor patient outcome (e.g. development of resistance to a treatment for CML, and / or progression to blast crisis phase). In some embodiments, the methods comprise evaluating a cancer to determine whether it comprises one of more given cell types / subtypes and / or the number / proportion of cells of a given cell type / subtype (e.g. megakaryocytic progenitor cells, lymphoid progenitor cells, inflamed HSPCs, inflamed lymphoid progenitor cells, adaptive NK cells).

[0119] Aspects and embodiments of the present disclosure also comprise selecting a subject for therapeutic or prophylactic intervention in accordance with the present disclosure. A subject having a cancer which is identified following such analysis to be a cancer comprising hallmark features indicative of poor patient outcome (e.g. development of resistance to a treatment for CML, and / or progression to blast crisis phase) may be selected for ‘aggressive’ therapeutic / prophylactic intervention. A subject having a cancer, which is identified following such analysis to be a cancer comprising hallmark features indicative of poor patient outcome (e.g. development of resistance to a treatment for CML, and / or progression to blast crisis phase) following administration of a given therapy (e.g. a first-generation TKI or a second-generation TKI), may be selected for ‘aggressive’ therapeutic / prophylactic intervention as described herein.

[0120] The present disclosure provides a method for predicting whether a subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. The present disclosure provides a method for determining whether a subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. The present disclosure provides, a method of identifying whether a CML patient is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0121] A method according to the present disclosure may be performed concurrently or subsequently to a CML diagnosis. That is, a method according to the present disclosure may be performed at the same time a subject is diagnosed with CML or following a subject’s CML diagnosis. A method according to the present disclosure may be performed within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks following a subject’s CML diagnosis. A method according to the present disclosure may be performed on a sample obtained from a subject before, at the same time as, or after a CML diagnosis. A method according to the present disclosure may be performed on a sample obtained from a subject within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks following a subject’s CML diagnosis. A method according to the present disclosure may be performed on a sample obtained from a subject who has CML or has been diagnosed with CML and has not received treatment for CML.

[0122] A method according to the present disclosure may be performed concurrently or subsequently to the administration of a treatment for CML to a subject. A method according to the present disclosure may be performed on a sample obtained from a subject who has CML or has been diagnosed with CML, and has received treatment for CML.

[0123] In some embodiments, the methods comprise determining one or more of:

[0124] (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs),

[0125] (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs,

[0126] (iii) the presence or absence of inflamed HSPCs, and

[0127] (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject.

[0128] In some embodiments, the methods comprise determining one or more of:

[0129] (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or hematopoietic cells,

[0130] (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs,

[0131] (iii) the presence or absence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and

[0132] (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject.

[0133] In some embodiments, the method comprises determining two or more (e.g. 2, 3, 4) of (i), (ii), (iii) and (iv) in a sample obtained from the subject. In some embodiments, the method comprises determining each of

[0134] (i), (ii), (iii) and (iv) in a sample obtained from the subject.

[0135] In some embodiments:

[0136] (i) an increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML,

[0137] (ii) an increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML,

[0138] (iii) the presence of inflamed HSPCs, and / or

[0139] (iv) the presence of inflamed lymphoid progenitor cells, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments:

[0140] (i) an increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently respond to treatment for CML,

[0141] (ii) an increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML,

[0142] (iii) the presence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and / or

[0143] (iv) the presence of inflamed lymphoid progenitor cells (e.g. the presence of inflamed lymphoid progenitor cells among CD35+ lin- HSPCs), indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0144] In some embodiments, determining two or more (e.g. 2, 3, 4) of (i), (ii), (iii) and (iv) indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, determining each of (i), (ii), (iii) and (iv) indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0145] In some embodiments, the methods further comprise determining:

[0146] (v) the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject.

[0147] In some embodiments:

[0148] (v) a reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion in chronic phase CML patients who subsequently respond to treatment for CML, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0149] In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:

[0150] (i) a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3%,

[0151] (ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs equal to or greater than 5%,

[0152] (iii) an increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently respond to treatment for CML,

[0153] (iv) an increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0154] (v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 51 %. In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises two or more (e.g. 2, 3, 4, 5) of (i), (ii), (iii), (iv) and (v). In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises each of (i), (ii), (iii) and (iv). In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises each of (i), (ii), (iii), (iv) and (v).

[0155] In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises: (iv) an increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in healthy subjects.

[0156] In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:

[0157] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently progress to blast crisis phase;

[0158] (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;

[0159] (iii) a similar or increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,

[0160] (iv) a similar or increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0161] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16br'9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16br'9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0162] In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:

[0163] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently progress to blast crisis phase;

[0164] (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase; (iii) a similar or increased number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive relative to the average number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,

[0165] (iv) a similar or increased number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0166] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0167] In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises two or more (e.g. 2, 3, 4, 5) of (i), (ii), (iii), (iv) and (v). In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises each of (i), (ii), (iii) and (iv). In some embodiments, a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises each of (i), (ii), (iii), (iv) and (v).

[0168] In some embodiments, a number / proportion which is ‘similar’ to a reference number / proportion may be >0.5 times and <2 times, e.g. one of >0.55 times and <1.9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1.3 times, >0.9 times and <1 .2 times or >0.95 times and <1 .1 times the reference number / proportion. In some embodiments, a number / proportion which is ‘increased’ relative to a reference number / proportion may be more than 1 times, e.g. >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the reference number / proportion. In some embodiments, a number / proportion which is ‘reduced’ relative to a reference number / proportion may be less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the reference number / proportion.

[0169] As used herein, a ‘chronic phase CML patient who subsequently responds to treatment for CML’ refers to a chronic phase CML patient who exhibits a reduction in the symptoms of CML, a reduction in a correlate of the severity / activity of CML (e.g. a reduction or maintenance of the % blast cells in the bone marrow and / or peripheral blood), or prevention of progression of CML, with treatment for CML. In some embodiments, a chronic phase CML patient who subsequently responds to treatment for CML may achieve a major molecular response or a deep molecular response with treatment for CML. Treatment response is principally monitored at the molecular level by quantitative measurement of BCR-ABL1 transcripts. Molecular response may be assessed e.g. using the guidelines set out in Hochhaus et al., Leukemia (2020) 34:966-984, which is hereby incorporated by reference in its entirety. This may be by assessing the ratio of BCR-ABL1 transcripts to ABL1 transcripts, e.g. using quantitative PCR. A major molecular response (MMR) is defined as <0.1% BCR-ABL1 on the international reporting scale (IS). A deep molecular response (DMR) is defined as <0.01% BCR-ABL1 on the international reporting scale (IS) (Branford, Hematology Am Soc Hematol Educ Program (2016) 1 :156-163, which is hereby incorporated by reference in its entirety). In some embodiments, chronic phase CML patients who subsequently respond to treatment for CML may be the ‘CPResp’ cohort as described in Examples 1 and 3 herein.

[0170] As used herein, a ‘chronic phase CML patient who subsequently progresses to blast crisis phase’ refers to a chronic phase CML patient who subsequently exhibits symptoms of blast crisis phase and / or is diagnosed with blast crisis phase. In some embodiments, blast crisis phase is identified as a patient having >30% blast cells within blood and / or bone marrow cells, and / or having extramedullary infiltrates of leukemic cells. A chronic phase CML patient who subsequently progresses to blast crisis phase may undergo blast crisis transformation after months or years (e.g. 3, 6, 9, 12, 24, 36, 48, 72, or 96 months) in chronic phase CML. In some embodiments, chronic phase CML patients who subsequently progresses to blast crisis phase’ may be the ‘CPMBC’ and / or ‘CPLBC’ cohort as described in Examples 1 and 3 herein.

[0171] In some embodiments, the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) is similar / increased / reduced relative to the average number / proportion of megakaryocytic progenitor cells among HSPCs in a reference population. In some embodiments, the number / proportion of megakaryocytic progenitor cells among CD45+ hematopoietic cells is similar / increased / reduced relative to the average number / proportion of megakaryocytic progenitor cells among CD45+ hematopoietic cells in a reference population. In some embodiments the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is similar / increased / reduced relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in a reference population. In some embodiments, the number / proportion of HSPCs that are pSTATI -positive is similar / increased / reduced relative to the average number / proportion of HSPCs that are pSTATI - positive in a reference population. In some embodiments, the number / proportion of megakaryocytic progenitors that are pSTATI -positive is similar / increased / reduced relative to the average number / proportion of megakaryocytic progenitors that are pSTATI -positive in a reference population. In some embodiments, the number / proportion of lymphoid progenitor cells that are I FITM1 -positive is similar / increased / reduced relative to the average number / proportion of lymphoid progenitor cells that are IFITM1 -positive in a reference population. In some embodiments, the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells is similar / increased / reduced relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in a reference population.

[0172] It will be appreciated that the number proportion of a given cell type / subtype in a sample obtained from the subject is similar / increased / reduced relative to the average number / proportion of a given cell type / subtype in a comparable sample (e.g. a sample of the same kind, e.g. obtained from the same fluid, tissue, organ etc., and / or obtained at the same stage of CML progression e.g. newly diagnosed, chronic phase) obtained from individuals in the reference population.

[0173] In some embodiments, the reference population is individuals with chronic phase CML who subsequently respond to treatment for CML. In some embodiments, the reference population is individuals with chronic phase CML who subsequently achieve a major molecular response (MMR) or a deep molecular response (DMR). In some embodiments, the reference population is individuals with chronic phase CML who subsequently have optimal response to treatment with a tyrosine kinase inhibitor. In some embodiments, individuals are newly diagnosed as having chronic phase CML (e.g. diagnosed in the preceding 1 , 2, 3, 4, 5, 6, 7, or 8 weeks). As used herein, ‘optimal response to treatment with a tyrosine kinase inhibitor’ refers to BCR-ABL1 expression of < 10% following 3 months of treatment, < 1% following 6 months of treatment, and / or < 0.1% following 12 months of treatment, using measurement of BCR-ABL / transcripts on the International Scale, as described in Hochhaus et al., Leukemia (2020) 34:966-894 and Hughes et al., Blood (2006) 108(1):28-37, which are hereby incorporated by reference in their entirety. In some embodiments, the reference population is the ‘CPResp’ cohort as described in Examples 1 and 3 herein.

[0174] In some embodiments, the reference population is individuals with chronic phase CML who subsequently progress to blast crisis phase. In some embodiments, individuals are newly diagnosed as having chronic phase CML (e.g. diagnosed in the preceding 1 , 2, 3, 4, 5, 6, 7, or 8 weeks). In some embodiments, the reference population is the ‘CPMBC’ and / or ‘CPLBC’ cohort as described in Examples 1 and 3 herein.

[0175] It will be appreciated that the average number / proportion of a given cell type / sub-type is that of the individuals of the reference population while they are in the chronic phase of CML. For example, the average number / proportion of a given cell type / sub-type prior to their treatment for CML or progression to blast crisis phase.

[0176] As used herein, the ‘average’ refers to the mean or median value. For example, the average number / proportion of megakaryocytic progenitor cells among HSPCs in a reference population, would be the mean or median value of the number / proportion of megakaryocytic progenitor cells among HSPCs of the individuals within the reference population.

[0177] In some embodiments, a method according to the present disclosure comprises detecting megakaryocytic progenitor cells. Megakaryocytic progenitor cells are derived from hematopoietic stem cells in the bone marrow. Megakaryocytic progenitor cells differentiate into megakaryocytes, which produce platelets (also known as thrombocytes).

[0178] In some embodiments, a method according to the present disclosure comprises determining the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), in a sample (e.g. a sample obtained from a subject). In some embodiments, a method according to the present disclosure comprises determining the number / proportion of megakaryocytic progenitor cells among CD45+ hematopoietic cells, in a sample (e.g. a sample obtained from a subject). In some embodiments, the megakaryocytic progenitor cells express CD42A. In some embodiments, the megakaryocytic progenitor cells express CD42A, and CD45 and / or CD34. In some embodiments, the megakaryocytic progenitor cells express CD42A, and CD45 and CD34. In some embodiments, the megakaryocytic progenitor cells do not express lineage markers (e.g. CD3 / 14 / 16 / 19). In some embodiments, the megakaryocytic progenitor cells express CD42A, CD45 and CD34, and do not express lineage markers (e.g. CD3 / 14 / 16 / 19). In some embodiments, the megakaryocytic progenitor cells are CD42A+ ( / .e. CD42A+ megakaryocytic progenitor cells). In some embodiments, the megakaryocytic progenitor cells are CD42A+, CD45+ and CD34+. In some embodiments, the megakaryocytic progenitor cells are Lin-, CD42A+, CD45+ and CD34+.

[0179] In some embodiments, a method according to the present disclosure comprises determining the number / proportion of CD42A+ cells among lin- ( / .e. CD3- / CD14- / CD16- / CD19-), CD45+and CD34+ cells.

[0180] In some embodiments, a method according to the present disclosure comprises determining the proportion of megakaryocytic progenitor cells among HSPCs in a sample (e.g. in a sample obtained from a subject). In some embodiments, according to the present disclosure a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3% indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, according to the present disclosure a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3.4% indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, according to the present disclosure a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 1%, e.g. >1.1%, >1.2%, >1.3%, >1.4%, >1.5%, >1.6%, >1.7%, >1.8%, >1.9%, >2%, >2.1%, >2.2%, >2.3%, >2.4%, >2.5%, >2.6%, >2.7%, >2.8%, >2.9%, >3%, >3.1%, >3.2%, >3.3%, >3.4%, >3.5%, >3.6%, >3.7%, >3.8%, >3.9%, >4%, >4.1%, >4.2%, >4.3%, >4.4%, >4.5%, >4.6%, >4.7%, >4.8%, >4.9%, >5%, >5.1%, >5.2%, >5.3%, >5.4%, >5.5%, >5.6%, >5.7%, >5.8%, >5.9%, 6%, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0181] In some embodiments, a method according to the present disclosure comprises determining the proportion of CD42A+ cells among lin-, CD45+ and CD34+ cells in a sample (e.g. in a sample obtained from a subject). In some embodiments, according to the present disclosure a proportion of CD42A+ cells among lin-, CD45+ and CD34+ cells equal to or greater than 3% indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase

[0182] In some embodiments, a method according to the present disclosure comprises detecting lymphoid progenitor cells. Lymphoid progenitor cells are hematopoietic stem cells that are committed to differentiate into T cells, B cells, or Natural Killer (NK) cells.

[0183] In some embodiments, a method according to the present disclosure comprises determining the number / proportion of lymphoid progenitor cells among lin-, CD45+, CD34+ and CD38+ cells in a sample (e.g. a sample obtained from a subject). In some embodiments, the lymphoid progenitor cells express CD19 and / or CD10. In some embodiments, the lymphoid progenitor cells express CD19 and CD10. In some embodiments, the lymphoid progenitor cells express CD19 and / or CD10, and one or more of CD45, CD34 and CD38. In some embodiments, the lymphoid progenitor cells express CD19 and CD10, and one or more of CD45, CD34, and CD38. In some embodiments, the lymphoid progenitor cells express CD19, CD10, CD45, CD34 and CD38. In some embodiments, the lymphoid progenitor cells do not express lineage markers (e.g. CD3 / CD14 / CD16). In some embodiments, the lymphoid progenitor cells express CD19, CD10, CD45, CD34 and CD38, and do not express lineage markers (e.g. CD3 / CD14 / CD16).

[0184] In some embodiments, the lymphoid progenitor cells are CD19+ and / or CD10+. In some embodiments, the lymphoid progenitor cells are CD19+ and CD10+ ( / .e. CD19+ CD10+ lymphoid progenitor cells). In some embodiments, the lymphoid progenitor cells are CD19+ and / or CD10+, and one or more of CD45+, CD34+ and CD38+. In some embodiments, the lymphoid progenitor cells are CD19+ and CD10+, and one or more of CD45+, CD34+ and CD38+. In some embodiments, the lymphoid progenitor cells are CD19+, CD10+, CD45+, CD34+ and CD38+. In some embodiments, the lymphoid progenitor cells are lin- (e.g. CD3 / CD14 / CD16), CD19+, CD10+, CD45+, CD34+ and CD38+.

[0185] In some embodiments, a method according to the present disclosure comprises determining the number / proportion of CD19+ and CD10+ cells among lin- ( / .e. CD3- / CD14- / CD16-), CD45+, CD34+ and CD38+ cells.

[0186] In some embodiments, a method according to the present disclosure comprises determining the proportion of lymphoid progenitor cells among lin-, CD45+, CD34+ and CD38+ cells in a sample (e.g. in a sample obtained from a subject). In some embodiments, according to the present disclosure a proportion of lymphoid progenitor cells among lin-, CD45+, CD34+ and CD38+ cells equal to or greater than 5% indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, according to the present disclosure a proportion of lymphoid progenitor cells among lin-, CD45+, CD34+ and CD38+ cells equal to or greater than 4.7% (e.g. equal to or greater than 4.78%) indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, according to the present disclosure a proportion of lymphoid progenitor cells among lin-, CD45+, CD34+ and CD38+ cells equal to or greater than 3%, e.g. >3.1%, >3.2%, >3.3%, >3.4%, >3.5%, >3.6%, >3.7%, >3.8%, >3.9%, >4%, >4.1%, >4.2%, >4.3%, >4.4%, >4.5%, >4.6%, >4.7%, >4.8%, >4.9%, >5%, >5.1%, >5.2%, >5.3%, >5.4%, >5.5%, >5.6%, >5.7%, >5.8%, >5.9%, 6%, >6.1%, >6.2%, >6.3%, >6.4%, >6.5%, >6.6%, >6.7%, >6.8%, >6.9%, >7%, >7.1%, >7.2%, >7.3%, >7.4%, >7.5%, >7.6%, >7.7%, >7.8%, >7.9%, >8%, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0187] In some embodiments, a method according to the present disclosure comprises determining the proportion of CD19+ and CD10+ cells among lin-, CD45+, CD34+ and CD38+ cells in a sample (e.g. in a sample obtained from a subject). In some embodiments, according to the present disclosure a proportion of CD19+ and CD10+ cells among lin-, CD45+, CD34+ and CD38+ cells equal to or greater than 5% indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0188] In some embodiments, a method according to the present disclosure comprises detecting HSPC inflammation in a sample (e.g. a sample obtained from a subject). In some embodiments, a method according to the present disclosure comprises determining the presence or absence of inflamed HSPCs in a sample (e.g. a sample obtained from a subject).

[0189] Hematopoietic stem and progenitor cells (HSPCs) are pluripotent cells that generate erythroid, lymphoid and myeloid lineages. In some embodiments, HSPCs express CD45 and CD34. In some embodiments, HSPCs do not express lineage markers. In some embodiments, HSPCs express CD45 and CD34, and do not express lineage markers. In some embodiments, HSPCs are CD45+ and CD34+. In some embodiments, HSPCs are lin-, CD45+ and CD34+.

[0190] As used herein, an ‘inflamed HSPC’ refers to a HSPC in which one or more pro-inflammatory signalling pathways (e.g. the IFNy pathway) are activated. An ‘inflamed HSPC’ may also be referred to as a HSPC in an inflammatory state or in a state of inflammation. In some embodiments, an ‘inflamed HSPC’ can be identified by measuring the presence of (or an increase in) a correlate of a pro-inflammatory signalling pathway. For example, by detecting phosphorylated STAT 1 as a correlate of IFNy signalling.

[0191] In some embodiments, the presence of inflamed HSPCs in a sample is indicated by an elevated number / proportion of HSPCs expressing pSTATI and / or an elevated number / proportion of HSPCs showing elevated expression of pSTATI . In some embodiments, a method according to the present disclosure comprises determining the number / proportion of HSPCs expressing pSTATI and / or the number / proportion of HSPCs showing elevated expression of pSTATI . In some embodiments, a method according to the present disclosure comprises determining the number / proportion of HSPCs expressing pSTATI and / or the number / proportion of HSPCs showing elevated expression of pSTATI among lin- CD45+ CD34+ cells.

[0192] In some embodiments, an increased number / proportion of HSPCs expressing pSTATI and / or an increased number / proportion of HSPCs showing elevated expression of pSTATI in a sample obtained from a subject, relative to the average number / proportion of HSPCs expressing pSTATI and / or the number / proportion of HSPCs showing elevated expression of pSTATI in a reference population (e.g. chronic phase CML patients who subsequently respond to treatment for CML), indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, a similar or increased number / proportion of HSPCs expressing pSTAT 1 and / or a similar or increased number / proportion of HSPCs showing elevated expression of pSTATI in a sample obtained from a subject, relative to the average number / proportion of HSPCs expressing pSTATI and / or the number / proportion of HSPCs showing elevated expression of pSTATI in a reference population (e.g. chronic phase CML patients who subsequently who subsequently progress to blast crisis phase), indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0193] In some embodiments, a method according to the present disclosure comprises detecting megakaryocytic progenitor cell inflammation in a sample (e.g. a sample obtained from a subject). Megakaryocytic progenitor cells are described hereinabove. As used an ‘inflamed megakaryocytic progenitor cell’ refers to a megakaryocytic progenitor cell in which one or more pro-inflammatory signalling pathways (e.g. the IFNy pathway) are activated. An ‘inflamed megakaryocytic progenitor cell’ may also be referred to as a megakaryocytic progenitor cell in an inflammatory state or in a state of inflammation. In some embodiments, an ‘inflamed megakaryocytic progenitor cell’ can be identified by measuring the presence of (or an increase in) a correlate of a pro- inflammatory signalling pathway. For example, by detecting phosphorylated STAT1 as a correlate of IFNy signalling.

[0194] In some embodiments, the presence of inflamed megakaryocytic progenitor cells in a sample is indicated by an elevated number / proportion of megakaryocytic progenitor cells expressing pSTATI and / or an elevated number / proportion of megakaryocytic progenitor cells showing elevated expression of pSTATI . In some embodiments, a method according to the present disclosure comprises determining the number / proportion of megakaryocytic progenitor cells expressing pSTATI and / or the number / proportion of megakaryocytic progenitor cells showing elevated expression of pSTATI . In some embodiments, a method according to the present disclosure comprises determining the number / proportion of megakaryocytic progenitor cells expressing pSTATI and / or the number / proportion of megakaryocytic progenitor cells showing elevated expression of pSTATI among lin- CD45+ CD34+ cells.

[0195] In some embodiments, an increased number / proportion of megakaryocytic progenitor cells expressing pSTATI and / or an increased number / proportion of megakaryocytic progenitor cells showing elevated expression of pSTATI in a sample obtained from a subject, relative to the average number / proportion of megakaryocytic progenitor cells expressing pSTATI and / or the number / proportion of megakaryocytic progenitor cells showing elevated expression of pSTATI in a reference population (e.g. chronic phase CML patients who subsequently respond to treatment for CML), indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, a similar or increased number / proportion of megakaryocytic progenitor cells expressing pSTATI and / or a similar or increased number / proportion of megakaryocytic progenitor cells showing elevated expression of pSTATI in a sample obtained from a subject, relative to the average number / proportion of megakaryocytic progenitor cells expressing pSTATI and / or the number / proportion of megakaryocytic progenitor cells showing elevated expression of pSTATI in a reference population (e.g. chronic phase CML patients who subsequently who subsequently progress to blast crisis phase), indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0196] In some embodiments, a method according to the present disclosure comprises detecting lymphoid progenitor cell inflammation in a sample (e.g. a sample obtained from a subject)

[0197] In some embodiments, a method according to the present disclosure comprises determining the presence or absence of inflamed lymphoid progenitor cells in a sample (e.g. a sample obtained from a subject).

[0198] As used herein, an ‘inflamed lymphoid progenitor cell’ refers to a lymphoid progenitor cell (e.g. a lymphoid progenitor cell as described herein) in which one or more pro-inflammatory signalling pathways (e.g. the I FNy pathway) are activated. An ‘inflamed lymphoid progenitor cell’ may also be referred to as a lymphoid progenitor cell in an inflammatory state or in a state of inflammation. In some embodiments, an ‘inflamed lymphoid progenitor cell’ can be identified by measuring the presence of (or an increase in) a correlate of a pro-inflammatory signalling pathway. For example, by detecting phosphorylated IFITM1 as a correlate of IFNy signalling. IFITM1 (interferon-induced transmembrane protein 1 or CD225) is an IFNy-induced inflammatory markers. It was found to be specifically upregulated within lymphoid progenitor cells in the blast crisis phase of CML.

[0199] In some embodiments, the presence of inflamed lymphoid progenitor cells in a sample is indicated by an elevated number / proportion of lymphoid progenitor cells expressing IFITM1 and / or an elevated number / proportion of lymphoid progenitor cells showing elevated expression of IFITM1. In some embodiments, a method according to the present disclosure comprises determining the number / proportion of lymphoid progenitor cells expressing IFITM1 and / or the number / proportion of lymphoid progenitor cells showing elevated expression of IFITM1. In some embodiments, a method according to the present disclosure comprises determining the number / proportion of lymphoid progenitor cells expressing IFITM1 and / or the number / proportion of lymphoid progenitor cells showing elevated expression of IFITM1 among CD19+ CD10+ lymphoid progenitor cells.

[0200] In some embodiments, an increased number / proportion of lymphoid progenitor cells expressing IFITM1 and / or an increased number / proportion of lymphoid progenitor cells showing elevated expression of IFITM1 in a sample obtained from a subject, relative to the average number / proportion of lymphoid progenitor cells expressing IFITM1 and / or the number / proportion of lymphoid progenitor cells showing elevated expression of IFITM1 in a reference population (e.g. chronic phase CML patients who subsequently respond to treatment for CML), indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, a similar or increased number / proportion of lymphoid progenitor cells expressing IFITM1 and / or a similar or increased number / proportion of lymphoid progenitor cells showing elevated expression of IFITM1 in a sample obtained from a subject, relative to the average number / proportion of lymphoid progenitor cells expressing IFITM1 and / or the number / proportion of lymphoid progenitor cells showing elevated expression of IFITM1 in a reference population (e.g. chronic phase CML patients who subsequently who subsequently progress to blast crisis phase), indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0201] In some embodiments, a method according to the present disclosure comprises detecting adaptive NK cells.

[0202] In humans, five NK cell subpopulations can be defined on the basis of the relative expression of the markers CD16 (or FcyRIIIA, low-affinity receptor for the Fc portion of immunoglobulin G) and CD56 (adhesion molecule mediating homotypic adhesion): (1) CD56bri9htCD16-, (2) CD56bri9htCD16dim, (3) CD56dimCD16-, (4) CD56dimCD16bri9ht, and (5) CD56- CD16bri9ht. These NK cell subpopulations are described in e.g. Poli et al., Immunology (2009), 126(4): 458-465, which is hereby incorporated by reference in its entirety. In some embodiments, a method according to the present disclosure comprises determining the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in a sample (e.g. a sample obtained from a subject). In some embodiments, the adaptive NK cells express NKG2C. In some embodiments, the adaptive NK cells express NKG2C, and one or more of CD45, CD56, and CD16. In some embodiments, the adaptive NK cells express NKG2C, CD45, CD56, and CD16. In some embodiments, the adaptive NK cells do not express lineage markers (e.g. CD3 / CD14 / CD19). In some embodiments, the adaptive NK cells express NKG2C, CD45, CD56, and CD16, and do not express lineage markers (e.g. CD3 / CD14 / CD19).

[0203] In some embodiments, the adaptive NK cells are NKG2C+ ( / .e. NKG2C+ adaptive NK cells). In some embodiments, the adaptive NK cells are NKG2C+, and one or more of CD45+, CD56+ (e.g. CD56dim), and CD16+ (e.g. CD16bri9ht). In some embodiments, the adaptive NK cells are NKG2C+, CD45+, CD56+ (e.g. CD56dim), and CD16+ (e.g. CD16bri9ht). In some embodiments, the adaptive NK cells are lin- (e.g. CD3- / CD14- / CD19-), NKG2C+, CD45+, CD56+ (e.g. CD56dim), and CD16+ (e.g. CD16brisht).

[0204] In some embodiments, a method according to the present disclosure comprises determining the proportion of NKG2C+ cells within a population of lin- (e.g. CD3 / CD14 / CD19), CD45+, CD56+ (e.g. CD56dim), and CD16+ (e.g. CD16bri9ht) cells.

[0205] In some embodiments, a method according to the present disclosure comprises determining the proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in a sample (e.g. in a sample obtained from a subject). In some embodiments, according to the present disclosure a proportion adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 51 % indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, according to the present disclosure a proportion adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 50.7% (e.g. less than or equal to 50.65%) indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, according to the present disclosure a proportion adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 60%, e.g. <59%, <58%, <57%, <56%, <55%, <54%, <53%, <52%, <51 %, <50%, <49%, <48%, <47%, <46%, <45%, <44%, <43%, <42%, <41 %, <40%, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0206] In some embodiments, a method according to the present disclosure comprises determining the proportion of NKG2C+ cells among lin- (e.g. CD3 / CD14 / CD19), CD45+, CD56+ (e.g. CD56dim), and CD16+ (e.g. CD16bri9ht) cells in a sample (e.g. in a sample obtained from a subject). In some embodiments, according to the present disclosure a proportion of NKG2C+ cells among lin- (e.g. CD3 / CD14 / CD19), CD45+, CD56+ (e.g. CD56dim), and CD16+ (e.g. CD16bri9ht) cells equal to or greater than 5% indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. Herein, a cell is determined to ‘express’ a given marker or be ‘positive’ for a given marker when it exhibits a positive or higher signal intensity (e.g. mean fluorescence intensity (MFI)) when stained with a detecting agent (e.g. an antibody specific to that marker) compared with a comparable cell stained with an isotype control, as assessed e.g. by flow cytometry or immunohistochemistry. By way of illustration, a cell which expresses CD34, is positive for CD34, or is CD34+, exhibits a positive or higher MFI when stained with an anti-CD34 antibody than a comparable cell stained with an isotype control, as assessed e.g. by flow cytometry. By way of another illustration, a cell which expresses CD34, is positive for CD34, or is CD34+, exhibits a positive or higher signal intensity when stained with an anti-CD34 antibody than a comparable cell stained with an isotype control, as assessed e.g. by immunohistochemistry.

[0207] Herein, a cell is determined to ‘not express’ a given marker or be ‘negative for a given marker when it does not exhibit a positive or higher signal intensity (e.g. mean fluorescence intensity (MFI)) when stained with a detecting agent (e.g. an antibody specific to that marker) compared with a comparable cell stained with an isotype control, as assessed e.g. by flow cytometry or immunohistochemistry. By way of illustration, a cell which does not express CD3, is negative for CD3, or is CD34-, does not exhibit a positive or higher MFI when stained with an anti-CD3 antibody than a comparable cell stained with an isotype control, as assessed e.g. by flow cytometry. By way of another illustration, a cell which does not express CD3, is negative for CD3, or is CD34-, does not exhibit a positive or higher signal intensity when stained with an anti-CD3 antibody than a comparable cell stained with an isotype control, as assessed e.g. by immunohistochemistry.

[0208] The number / proportion / presence / absence of cells of a given cell type / subtype described herein can be determined e.g. by analysis using antibodies for the detection of markers for the given cell type / subtype as described herein, permitting the identification of such cells from within heterogeneous cell populations. The number / proportion / presence / absence of cells of a given cell type / subtype described herein can be determined by analysis by an appropriate method, e.g. by flow cytometry (e.g. multicolour flow cytometry) or immunohistochemistry, using antibodies providing for the detection of the given cell type / subtype as described herein.

[0209] In some embodiments, cells of a given type / subtype are detected by cell surface markers ( / .e. markers present / expressed on the surface of the cell). In some embodiments, cells of a given type / subtype are detected using cell surface staining, e.g. cell surface antibody staining. In some embodiments, cells of a given type / subtype are detected by intracellular markers ( / .e. markers present / expressed in the cytosol of the cell). In some embodiments, cells of a given type / subtype are detected using intracellular staining, e.g. intracellular antibody staining. In some embodiments, cells of a given type / subtype are detected using a combination of cell surface markers and intracellular markers. In some embodiments, cells of a given type / subtype are detected using a combination of cell surface staining and intracellular staining.

[0210] In some embodiments, number / proportion / presence / absence of cells of a given cell type / subtype can be determined essentially as described in Example 1 herein. In some embodiments, a method according to the present disclosure comprises the detection of one or more markers indicative of cell type / subtype (e.g. cell surface markers and / or intracellular markers). In some embodiments, a method according to the present disclosure comprises determining the expression of one or more markers.

[0211] In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38, CD45RA, CD42A, CD10, CD90, CD123, pSTATI and IFITM1 . In some embodiments, a method according to the present disclosure further comprises determining the expression of one or more of CD71 , CD105, CD253A, CD56, NKG2A and / or NKG2C.

[0212] In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD14, CD16, CD34, CD38, CD42A, CD19, CD10, pSTATI and IFITM1 . In some embodiments, a method according to the present disclosure comprises determining the expression of CD45, CD3, CD14, CD16, CD34, CD38, CD42A, CD19, CD10, pSTATI and IFITM1. In some embodiments, a method according to the present disclosure further comprises determining the expression of NKG2C+ and / or CD56. In some embodiments, a method according to the present disclosure further comprises determining the expression of NKG2C+ and CD56.

[0213] In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38, CD45RA and CD42A. In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38 and CD42A. In some embodiments, a method according to the present disclosure comprises determining the expression of CD45, CD3, CD14, CD16, CD19, CD34, CD38 and CD42A.

[0214] In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD14, CD16, CD34, CD38, CD10 and CD19. In some embodiments, a method according to the present disclosure comprises determining the expression of CD45, CD3, CD14, CD16, CD34, CD38, CD10 and CD19.

[0215] In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38, CD90, CD45RA, CD123 and pSTATI (Ser727). In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD19, CD14, CD16, CD34, CD38 and pSTATI (Ser727). In some embodiments, a method according to the present disclosure comprises determining the expression of CD45, CD3, CD19, CD14, CD16, CD34, CD38 and pSTATI (Ser727).

[0216] In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD14, CD16, CD34, CD38, CD10, CD19 and IFITM1. In some embodiments, a method according to the present disclosure comprises determining the expression of CD45, CD3, CD14, CD16, CD34, CD38, CD10, CD19 and IFITM1. In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD14, CD19, CD34, CD71 , CD105, CD235A, CD56, CD16, NKG2A and NKG2C. In some embodiments, a method according to the present disclosure comprises determining the expression of one or more of CD45, CD3, CD14, CD19, CD56, CD16 and NKG2C. In some embodiments, a method according to the present disclosure comprises determining the expression of CD45, CD3, CD14, CD19, CD56, CD16 and NKG2C.

[0217] Antibodies suitable for use in the methods described herein (e.g. suitable for determining the expression of the markers described herein) are known to the skilled person.

[0218] In some embodiments, a method according to the present disclosure is for predicting whether a subject is at risk of developing resistance to a treatment for CML.

[0219] As used herein, a subject that has developed ‘resistance’ to a given treatment, has a cancer that is resistant / does not respond to the relevant treatment. A subject that has developed ‘resistance’ to a given treatment, may not display a partial or complete response to the relevant treatment. A cancer that is resistant / does not respond to a given treatment, may be a cancer in which growth / progression was not inhibited by the relevant treatment. A cancer that is resistant / does not respond to a given treatment, may be a cancer for which a subject receiving a given treatment for the cancer did not display a partial or complete response to the relevant treatment. Resistance may be primary or secondary resistance. Primary resistance refers to the lack of hematologic, cytogenetic, or molecular response to a drug in the early stages of treatment, whereas secondary resistance refers to the loss of response to a drug following an initial therapeutic response.

[0220] Resistance may be relative resistance or absolute resistance. That is, a method of the present disclosure may be a method for predicting whether a subject is at risk of developing relative and / or absolute resistance to a treatment for CML.

[0221] In some embodiments, the resistance is relative resistance. That is, a method of the present disclosure may be a method for predicting whether a subject is at risk of developing relative resistance to a treatment for CML. Patients who have relative resistance, have CML that is still dependent on BCR::ABL1 kinase activity. Patients who have relative resistance may eventually reach a treatment response associated with low long-term risk, but only after a delay compared to patients who achieve an optimal treatment response early and who may be eligible for treatment discontinuation. For patients with relative resistance, treatment with a TKI that is more potent than a first generation TKI may allow said patients to achieve improved responses. For example, treatment with a second-generation TKI, third-generation TKI, or STAMP inhibitor may allow patients with relative resistance to achieve improved responses.

[0222] In some embodiments, the resistance is absolute resistance. That is, a method of the present disclosure may be a method for predicting whether a subject is at risk of developing absolute resistance to a treatment for CML. Patients who have absolute resistance have CML comprising cancer cells which are BCR: :ABL1 -independent.

[0223] A ‘treatment for CML’ may be any treatment for CML, e.g. a treatment for CML as described herein. In some embodiments, the treatment for CML is a tyrosine kinase inhibitor. In some embodiments, the treatment for CML comprises a tyrosine kinase inhibitor. In some embodiments, the treatment for CML is one or more tyrosine kinase inhibitors.

[0224] In some embodiments, a method according to the present disclosure is for predicting whether a subject is at risk of developing resistance to treatment with a tyrosine kinase inhibitor. In some embodiments, a method according to the present disclosure is for predicting whether a subject is at risk of having a CML that does not respond to treatment with a tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is selected from one of: imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, radotinib, olverembatinib, flumatinib, asciminib, and TERN-701. In some embodiments, the tyrosine kinase inhibitor is a first-line TKI, a second-line TKI, and / or a third-line TKL In some embodiments, the TKI is a STAMP inhibitor. In some embodiments, the STAMP inhibitor is asciminib or TERN-701 . In some embodiments, the TKI is not a STAMP inhibitor.

[0225] In some embodiments, a method according to the present disclosure is for predicting whether a subject is at risk of progressing to blast crisis phase (e.g. blast crisis phase as defined herein).

[0226] In some embodiments, a method according to the present disclosure is for assessing / determining / monitoring the response of a subject to a therapeutic / prophylactic intervention as described herein.

[0227] Disclosed herein is a method of assessing / determining / monitoring a subject’s response to a treatment for chronic myelogenous leukaemia (CML), the method comprising:

[0228] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the number / proportion of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and (iv) the number / proportion of inflamed lymphoid progenitor cells, in a sample obtained from the subject at a first timepoint; and

[0229] (b) determining one or more of: (i), (ii), (iii), and / or (iv) in a sample obtained from the subject at a subsequent timepoint.

[0230] In some embodiments, the method comprises determining each of (i), (ii), (iii), and (iv) in the sample obtained from the subject at the first timepoint. In some embodiments, the method comprises determining each of (i), (ii), (iii), and (iv) in the sample obtained from the subject at the subsequent timepoint. In some embodiments, the method comprises determining each of (i), (ii), (iii), and (iv) in the sample obtained from the subject at the first timepoint, and each of (i), (ii), (iii), and (iv) in the sample obtained from the subject at the subsequent timepoint. In some embodiments, the method further comprises:

[0231] (a) determining (v) the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject at the first timepoint; and

[0232] (b) determining (v) in a sample obtained from the subject at a subsequent timepoint.

[0233] In some embodiments,

[0234] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells,

[0235] (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs,

[0236] (iii) a similar or increased number / proportion of inflamed HSPCs and / or inflamed megakaryocytic progenitors,

[0237] (iv) a similar or increased number / proportion of inflamed lymphoid progenitor cells, and / or

[0238] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells, determined in the sample obtained from the subject at the subsequent timepoint compared with the number / proportion of (i), (ii), (iii), (iv), and / or (v) determined in the sample obtained from the subject the first timepoint indicates that the subject has a poor response, or no response, to the treatment for CML.

[0239] In some embodiments,

[0240] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells,

[0241] (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs,

[0242] (iii) a similar or increased number / proportion of inflamed HSPCs and / or inflamed megakaryocytic progenitors,

[0243] (iv) a similar or increased number / proportion of inflamed lymphoid progenitor cells, and / or

[0244] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16brishtNK cells, determined in the sample obtained from the subject at the subsequent timepoint compared with the number / proportion of (i), (ii), (iii), (iv), and / or (v) determined in the sample obtained from the subject the first timepoint indicates that the subject has developed resistance to the treatment for CML.

[0245] In some embodiments, a subject identified as having a poor response / no response / developed resistance to the treatment for CML may be selected for further treatment with a more aggressive / potent treatment for CML. For example, a subject identified as having a poor response / no response / developed resistance to a first-generation TKI may be selected for further treatment comprising one or more of a second- generation TKI, a third-generation TKI, a STAMP inhibitor, a combination of a TKI and chemotherapy, a combination of a TKI and one or more steroids, or a stem cell transplant. For example, a subject identified as having a poor response / no response / developed resistance to a second-generation TKI may be selected for further treatment comprising one or more of a third-generation TKI, a STAMP inhibitor, a combination of a TKI and chemotherapy, a combination of a TKI and one or more steroids, or a stem cell transplant. In some embodiments, the first timepoint is concurrent or subsequent to a CML diagnosis. That is, the first timepoint may be at the same time a subject is diagnosed with CML or following a subject’s CML diagnosis. In some embodiments, the first timepoint is within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks following a subject’s CML diagnosis. In some embodiments, the first timepoint is prior to the administration of a treatment for CML.

[0246] In some embodiments, the subsequent timepoint is 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, or 26 weeks after the first timepoint. In some embodiments, the subsequent timepoint is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 months after the first timepoint. In some embodiments, the subsequent timepoint is 1 , 2, 3, 4, or 5 years after the first timepoint.

[0247] In some embodiments, the subsequent timepoint is after the subject has received treatment for CML. In some embodiments, the subsequent timepoint is concurrent with the subject receiving treatment for CML. In some embodiments, the subsequent timepoint is 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, or 26 weeks after administration of a treatment for CML (e.g. after administration of the first dose of a given therapeutic agent). In some embodiments, the subsequent timepoint is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 months after administration of a treatment for CML (e.g. after administration of the first dose of a given therapeutic agent). In some embodiments, the subsequent timepoint is 1 , 2, 3, 4, or 5 years after administration of a treatment for CML (e.g. after administration of the first dose of a given therapeutic agent).

[0248] For example, the first timepoint may be concurrent with / at the time a subject is diagnoses with CML and / or prior to the administration to the subject of a treatment for CML; and the subsequent timepoint may be following administration of a treatment for CML to the subject. That is, the separation of the first timepoint and subsequent timepoint allows for assessing / determining / monitoring the response of the subject to the treatment for CML. It will be appreciated that a treatment for CML may comprise administration of multiple doses of a given agent (e.g. a TKI) over a period of time. In some embodiments, the subsequent timepoint may be following one or more doses of a given agent.

[0249] Therapeutic and prophylactic intervention

[0250] The present disclosure provides methods for the treatment and / or prevention of chronic myelogenous leukaemia (CML).

[0251] The methods may be effective to reduce the development or progression of CML, alleviate the symptoms of CML or reduce the pathology of CML. The methods may be effective to prevent progression of CML, e.g. to prevent worsening of, or to slow the rate of development of, CML. In some embodiments, the methods may lead to an improvement in CML, e.g. a reduction in the symptoms of CML or reduction in some other correlate of the severity / activity of CML. In some embodiments, the methods may prevent development of CML to a later stage (e.g. blast crisis phase or metastasis). Treatments for CML include tyrosine kinase inhibitors (e.g. imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, radotinib, olverembatinib, flumatinib, asciminib, and TERN-701), chemotherapeutic agents (e.g. hydroxycarbamide, fludarabine, cytarabine, idarubicin, buslfan, cyclophosphamide, vincristine, omacetaxine, dexamethasone), interferon therapy (e.g. interferon-alpha), radiation therapy, stem cell therapy (e.g. stem cell transplant (e.g. autologous stem cell transplant, allogeneic stem cell transplant)), immunotherapy, or combinations thereof. Treatments for CML are reviewed in Helhmann, J Clin Med. (2020), 9(11):3671 , which is hereby incorporated by reference in its entirety.

[0252] Resistance may develop during treatment of CML. Resistance (e.g. TKI resistance) can be driven by BCR-ABL-dependent and independent mechanisms. BCR-ABL-dependent mechanisms include overexpression of the BCR-ABL gene and point mutations in the BCR-ABL kinase domain that prevent the binding of the drug (e.g. the TKI). BCR-ABL independent mechanisms include alterations in drug influx and efflux, and activation of BCR-ABL independent mechanisms (such as members of the Src family of kinases). Resistance to TKIs in CML is reviewed e.g in Alves et al., Cancers (Basel) (2021) 13(19):4820.

[0253] It may be appropriate to use different therapies for treatment of CML in different stages. For example, treatment of CML in the blast crisis phase may involve more aggressive therapy options. Treatment of blast crisis phase of CML is reviewed e.g. in Hehlmann, Blood (2012) 120(4):737, which is hereby incorporated by reference in its entirety.

[0254] Treatment of CML may comprise the use of tyrosine kinase inhibitors (TKIs). TKIs are small molecules that disrupt tyrosine kinases through different modes of action. TKIs may compete with the adenosine triphosphate-binding site of the catalytic domain of a tyrosine kinase, which inhibits autophosphorylation and activation of the tyrosine kinase, and prevents activation of downstream intracellular signalling pathways. Alternatively, TKIs may act as allosteric inhibitors in which the TKI binds to a site other than the active site of the tyrosine kinase, causing a conformational change of the enzyme. TKIs used in the treatment of CML may target BCR-ABL.

[0255] TKIs (e.g. TKIs targeting BCR-ABL) are often the first-line therapy for patients with CML. Imatinib is a first generation BCR-ABL TKI. Second generation BCR-ABL TKIs (which are more potent than imatinib) include nilotinib, dasatinib, bosutinib, and bafetinib. Ponatinib, is an example of a third generation BCR- ABL TKI, and is more potent than first and second generation BCR-ABL TKIs. Patients with CML may be treated with a first-line TKI, and then treated with a second-line TKI (for example, if the patient does not respond to treatment with the first-line TKI or becomes resistant to the first-line TKI). Patients with CML may then treated with a third-line TKI (for example, if the patient does not respond to treatment with the second-line TKI or becomes resistant to the second-line TKI). Known TKIs imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, radotinib, olverembatinib, flumatinib, asciminib, and TERN-701.

[0256] As used herein, a “first-generation TKI” may include imatinib. A “second-generation TKI” may include dasatinib, nilotinib, bosutinib, bafetinib, radotinib, or flumatinib. A “third-generation TKI” may include ponatinib or olverembatinib. As used herein, a “first-line TKI”, a “second-line TKI”, or a “third line TKI” may refer to a “first-generation TKI”, a “second-generation TKI”, and a “third-generation TKI”, respectively.

[0257] Treatment of CML may comprise the use of STAMP inhibitors. STAMP inhibitors (Specifically Targeting the ABL Myristoyl Pocket inhibitors) are a type of tyrosine kinase inhibitor. STAMP inhibitors specifically target the ABL myristoyl pocket, and therefore block BCR-ABL1 kinase activity via allosteric binding. Known STAMP inhibitors include asciminib (DrugBank Accession No. DB12597) and TERN-701 (also known as HS-10382, and as described in e.g. Parsons et al., Blood (2023) 142 (Supplement 1): 5757, which is hereby incorporated by reference in its entirety).

[0258] Treatment of CML may comprise the use of chemotherapeutic agents. Chemotherapeutic agents useful in the treatment of CML include hydroxycarbamide, fludarabine, cytarabine, idarubicin, busulfan, cyclophosphamide, vincristine, omacetaxine, dexamethasone. Chemotherapeutic agents may be used alone in combination with other CML treatments, e.g. TKIs.

[0259] Treatment of CML may comprise the use of stem cell therapy or a stem cell transplant. Stem cell transplantation may be used, e.g. for treatment of accelerated or blastic phase CML, or when a patient does not respond to a first line treatment e.g. TKI. The role of stem cell transplantation in the treatment of CML is reviewed e.g. in Barrett & Ito, Blood (2015) 125(21):3230-3235, which is hereby incorporated by reference in its entirety.

[0260] In some embodiments, a method according to the present disclosure is for predicting whether a subject is at risk of developing resistance to one or more treatments for CML (e.g. one or more treatments for CML as described herein).

[0261] In some embodiments, a method according to the present disclosure is for predicting whether a subject is at risk of developing resistance to a TKI. In some embodiments, a method according to the present disclosure is for predicting whether a subject is at risk of developing resistance to a TKI selected from: imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, radotinib, olverembatinib, flumatinib, asciminib, and TERN-701 . In some embodiments, a method according to the present disclosure is for predicting whether a subject is at risk of developing resistance to a first-line TKI, a second-line TKI, and / or a third- line TKI. In some embodiments, the TKI is a STAMP inhibitor. In some embodiments, the STAMP inhibitor is selected from: asciminib, and TERN-701 . In some embodiments, the TKI is not a STAMP inhibitor.

[0262] In some embodiments, methods disclosed herein comprise selecting a subject for treatment, wherein the treatment comprises one or more treatments for CML (e.g. one or more treatments for CML as described herein).

[0263] In some embodiments, methods disclosed herein comprise selecting a subject for treatment, wherein the treatment comprises a TKI. In some embodiments, methods disclosed herein comprise selecting a subject for treatment, wherein the treatment comprises a TKI selected from imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, radotinib, olverembatinib, flumatinib, asciminib, and TERN-701. In some embodiments, methods disclosed herein comprise selecting a subject for treatment, wherein the treatment comprises a first-generation TKI, a second-generation TKI, and / or a third-generation TKI. In some embodiments, the TKI is a STAMP inhibitor. In some embodiments, the STAMP inhibitor is selected from: asciminib, and TERN-701. In some embodiments, the TKI is not a STAMP inhibitor.

[0264] In some embodiments, methods disclosed herein comprise selecting a subject for treatment, wherein the treatment comprises a stem cell transplant.

[0265] The present disclosure provides a method for treating or preventing CML in a subject. It will be appreciated that the method may comprise administering any of the treatments / therapies for CML described herein.

[0266] In some embodiments, the subject has been predicted to be at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. In some embodiments, the subject has been predicted not to be at risk of developing resistance to a treatment for CML and / or progressing to clast crisis phase.

[0267] The present disclosure provides a method for treating or preventing CML in a subject. A subject may be selected / stratified for treatment with a given therapy. In some embodiments, a method for treating or preventing CML according to the present disclosure comprises selecting a patient for a given treatment as informed by whether a subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase (e.g. as predicted according to a method for predicting whether a subject is at risk of developing resistance to a treatment for CML and / or progressing to clast crisis phase according to the present disclosure). In some embodiments, a method for treating or preventing CML according to the present disclosure comprises determining if a subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase (e.g. according to a method of determining if a subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase according to the present disclosure). In some embodiments, a method for treating or preventing CML according to the present disclosure comprises analysing a sample obtained from the subject.

[0268] A subject (e.g. a subject with chronic phase or accelerated phase CML) that has been determined to be at risk of developing resistance to a treatment for CML and / or progressing to blast phase may be treated with a more “aggressive” (e.g. more potent or effective) therapy than a conventional first line therapy (e.g. a first-generation TKI or imatinib).

[0269] For example, a subject that has been determined to be at risk of developing resistance to a treatment for CML and / or progressing to blast phase may be treated with a treatment selected from a second- generation TKI (e.g. bosutinib, dasatinib, or nilotinib), a third-generation TKI (e.g. ponatinib), a STAMP inhibitor (e.g. asciminib), a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third-generation TKI) and chemotherapy (e.g. AML or ALL-type induction chemotherapy, or a chemotherapeutic agent as disclosed herein), a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third-generation TKI) and one or more steroids, or a stem cell transplant (e.g. allogeneic hematopoietic cell transplant).

[0270] By way of an illustrative example, a subject (e.g. a subject with chronic phase or accelerated phase CML) that has been determined to be at risk of developing resistance to a first-generation TKI (e.g. imatinib) may be treated with a second-generation TKI (e.g. bosutinib, dasatinib, or nilotinib), a third-generation TKI (e.g. ponatinib), a STAMP inhibitor (e.g. asciminib), a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third-generation TKI) and chemotherapy, a combination of a TKI (e.g. a first- generation TKI, a second-generation TKI, or a third-generation TKI) and one or more steroids, or a stem cell transplant (e.g. allogeneic hematopoietic cell transplant).

[0271] By way of another illustrative example, a subject (e.g. a subject with chronic phase or accelerated phase CML) that has been determined to be at risk of developing resistance to a second-generation TKI (e.g. bosutinib, dasatinib, or nilotinib) may be treated with an alternative second-generation TKI, a third- generation TKI (e.g. ponatinib), a STAMP inhibitor (e.g. asciminib), a combination of a TKI (e.g. a first- generation TKI, a second-generation TKI, or a third-generation TKI) and chemotherapy, a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third-generation TKI) and one or more steroids, or a stem cell transplant (e.g. allogeneic hematopoietic cell transplant). For example, a subject that has been determined to be at risk of developing resistance to bosutinib may be treated with dasatinib or nilotinib, or a third-generation TKI (e.g. ponatinib), a STAMP inhibitor (e.g. asciminib), a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third-generation TKI) and chemotherapy, a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third-generation TKI) and one or more steroids, or a stem cell transplant (e.g. allogeneic hematopoietic cell transplant). A subject that has been determined to be at risk of developing resistance to dasatinib may be treated with bosutinib or nilotinib, a third-generation TKI (e.g. ponatinib), a STAMP inhibitor (e.g. asciminib), a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third-generation TKI) and chemotherapy, a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third- generation TKI) and one or more steroids, or a stem cell transplant (e.g. allogeneic hematopoietic cell transplant). A subject that has been determined to be at risk of developing resistance to nilotinib may be treated with bosutinib or dasatinib, a third-generation TKI (e.g. ponatinib), a STAMP inhibitor (e.g. asciminib), a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third- generation TKI) and chemotherapy, a combination of a TKI (e.g. a first-generation TKI, a second- generation TKI, or a third-generation TKI) and one or more steroids, or a stem cell transplant (e.g. allogeneic hematopoietic cell transplant).

[0272] A subject (e.g. a subject with chronic phase or accelerated phase CML) that has been determined not to be at risk of developing resistance to a treatment for CML and / or progressing to blast phase may be treated with a conventional first-line therapy. Conventional first line therapies for a subject with CML (e.g. chronic phase CML) may include a first-generation TKI (e.g. imatinib). The selection of a first-line TKI may be based on factors such as risk score, toxicity profile, age of the subject, the ability to tolerate therapy, and the presence of co-morbid conditions. First line therapies for a subject with CML (e.g. chronic phase CML) may include a second-generation TKI (e.g. bosutinib, dasatinib, or nilotinib). Aspects and embodiments of the present disclosure also comprise selecting a subject for therapeutic or prophylactic intervention in accordance with the present disclosure.

[0273] In some embodiments, the methods comprise selecting a subject for treatment with a more “aggressive” (e.g. more potent or effective) therapy than a conventional first line therapy, as disclosed herein. In some embodiments, the methods comprise identifying a subject having a poor response / no response / developed resistance to a given treatment for CML, and selecting said subject for treatment with a more “aggressive” (e.g. more potent or effective) therapy than the given therapy.

[0274] In some embodiments, the methods comprise:

[0275] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;

[0276] (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) is increased relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is increased relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML, (iii) inflamed HSPCs are present, and (iv) inflamed lymphoid progenitor cells are present.

[0277] In some embodiments, the methods comprise:

[0278] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;

[0279] (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells is increased relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently respond to treatment for CML, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is increased relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML, (iii) inflamed HSPCs and / or inflamed megakaryocytic progenitors are present, and (iv) inflamed lymphoid progenitor cells are present.

[0280] In some embodiments, the methods further comprise: (c) administering a more aggressive (e.g. more potent or effective) therapy than a conventional first line therapy as disclosed herein to a subject selected for treatment in step (b). In some embodiments, the more aggressive therapy is selected from the list consisting of a second-generation TKI (e.g. bosutinib, dasatinib, or nilotinib), a third-generation or TKI (e.g. ponatinib), a STAMP inhibitor (e.g. asciminib), a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third- generation TKI) and chemotherapy (e.g. AML or ALL-type induction chemotherapy, or a chemotherapeutic agent as disclosed herein), a combination of a TKI (e.g. a first-generation TKI, a second-generation TKI, or a third-generation TKI) and one or more steroids, or a stem cell transplant (e.g. allogeneic hematopoietic cell transplant).

[0281] In some embodiments, step (a) of the methods further comprise determining the number / proportion of adaptive NK cells among CD56dimCD16br'9htNK cells in the sample obtained from the subject; and step (b) of the methods further comprise selecting the subject for treatment if the number / proportion of adaptive NK cells among CD56dimCD16br'9htNK cells is reduced relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML.

[0282] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0283] (i) a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3%,

[0284] (ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs equal to or greater than 5%,

[0285] (iii) an increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently respond to treatment for CML,

[0286] (iv) an increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0287] (v) a proportion of adaptive NK cells among CD56dimCD16br'9htNK cells less than or equal to 51 %.

[0288] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0289] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently progress to blast crisis phase;

[0290] (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;

[0291] (iii) a similar or increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase, (iv) a similar or increased number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0292] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0293] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0294] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently progress to blast crisis phase;

[0295] (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;

[0296] (iii) a similar or increased number / proportion of HSPCs and / or megakaryocytic progenitor cells that are pSTATI -positive relative to the average number / proportion of HSPCs megakaryocytic progenitor cells that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,

[0297] (iv) a similar or increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0298] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0299] In some embodiments, a method comprises selecting a subject for treatment with a conventional first line therapy (e.g. a first-generation or second-generation TKI).

[0300] In some embodiments, the methods comprise:

[0301] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;

[0302] (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) is reduced relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently progress to blast crisis phase, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is reduced relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase, (iii) inflamed HSPCs are not present, and (iv) inflamed lymphoid progenitor cells are not present.

[0303] In some embodiments, the methods comprise:

[0304] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs and / or inflamed megakaryocytic progenitor cells, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;

[0305] (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells is reduced relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently progress to blast crisis phase, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is reduced relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase, (iii) inflamed HSPCs inflamed megakaryocytic progenitor cells are not present, and (iv) inflamed lymphoid progenitor cells are not present.

[0306] In some embodiments, the treatment comprises one or more of a first-generation TKI or a second- generation TKL In some embodiments, the methods further comprise:

[0307] (c) administering the treatment to a subject selected for treatment in step (b).

[0308] In some embodiments, step (a) of the methods further comprise determining the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject; and step (b) of the methods further comprise selecting the subject for treatment if the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells is increased relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0309] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0310] (i) a proportion of megakaryocytic progenitor cells among HSPCs less than or equal to 3%,

[0311] (ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs less than or equal to than 5%,

[0312] (iii) a reduced number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently progress to blast crisis phase,

[0313] (iv) a reduced number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0314] (v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells equal to or greater than 51 %. In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0315] (i) a similar or reduced number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML;

[0316] (ii) a similar or reduced number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML;

[0317] (iii) a similar or reduced number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently respond to treatment for CML,

[0318] (iv) a similar or reduced number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0319] (v) a similar or increased number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML.

[0320] In some embodiments, a sample obtained from a subject selected for treatment comprises one or more of:

[0321] (i) a similar or reduced number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) CD45+ hematopoietic cells in chronic phase CML patients who subsequently respond to treatment for CML;

[0322] (ii) a similar or reduced number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML;

[0323] (iii) a similar or reduced number / proportion of HSPCs and / or megakaryocytic progenitor cells that are pSTATI -positive relative to the average number / proportion of HSPCs and / or megakaryocytic progenitor cells that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently respond to treatment for CML,

[0324] (iv) a similar or reduced number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0325] (v) a similar or increased number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML. Determining the number / proportion of a given cell type / sub-type in any of the above embodiments, may comprise analysis using antibodies for the detection of markers for the given cell type / subtype as described herein, permitting the identification of such cells from within heterogeneous cell populations. The number / proportion / presence / absence of cells of a given cell type / subtype described herein can be determined by analysis by an appropriate method, e.g. by flow cytometry (e.g. multicolour flow cytometry) or immunohistochemistry, using antibodies providing for the detection of the given cell type / subtype as described herein.

[0326] It will be appreciated that the sample may be taken from any tissue or bodily fluid. In some embodiments, the sample comprises or is derived from a quantity of blood (e.g. peripheral blood) or from bone marrow. In some embodiments, the sample comprises bone marrow mononuclear cells (BM-MNCs) or peripheral blood mononuclear cells (PB-MNCs).

[0327] Also provided is a method for improving the prognosis of CML in a subject, e.g. a subject determined to be at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0328] Also provided is a method of conditioning a CML patient (e.g. a patient determined to be at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase) for treatment with a TKI (e.g. a first-generation TKI or a second-generation TKI).

[0329] In some embodiments, the method of improving prognosis / conditioning comprises (i) reducing the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) reducing the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) reducing the number of inflamed HSPCs, and / or (iv) reducing the number of inflamed lymphoid progenitor cells in the subject / patient.

[0330] In some embodiments, the method of improving prognosis / conditioning comprises (i) reducing the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) reducing the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) reducing the number of inflamed HSPCs and / or inflamed megakaryocytic progenitor cells, and / or (iv) reducing the number of inflamed lymphoid progenitor cells in the subject / patient.

[0331] It will be appreciated that any treatment capable of (i) reducing the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) reducing the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) reducing the number of inflamed HSPCs, and / or (iv) reducing the number of inflamed lymphoid progenitor cells in a subject would find utility in the methods. It will also be appreciated that any treatment capable of (i) reducing the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) reducing the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) reducing the number of inflamed HSPCs and / or inflamed megakaryocytic progenitor cells, and / or (iv) reducing the number of inflamed lymphoid progenitor cells in a subject would find utility in the methods. In some embodiments, the method comprises killing / reducing proliferation of a given cell type (e.g. megakaryocytic progenitor cells, lymphoid progenitor cells). In some embodiments, the method comprises reducing inflammation (e.g. inhibiting one or more pro-inflammatory signalling pathways) within a given cell type (e.g. HSPCs, megakaryocytic progenitor cells, lymphoid progenitor cells).

[0332] In some embodiments, the method of improving prognosis / conditioning comprises administering an agent capable of targeting a given cell population. In some embodiments, the method of improving prognosis / conditioning comprises administering an agent capable of killing / reducing proliferation of a given cell type.

[0333] In some embodiments, the method comprises administration of an agent capable of binding to one or more markers of a given cell type (e.g. megakaryocytic progenitor cells, lymphoid progenitor cells, inflamed HSPCs, inflamed lymphoid progenitor cells). In some embodiments, the method comprises administration of an agent capable of binding to one or more markers selected from: CD42A, CD19, CD10, CD45, CD34, and CD38. In some embodiments, the agent is capable of binding to CD42A. In some embodiments, the agent is capable of binding to CD19 and / or CD10.

[0334] In some embodiments, the agent is an antibody or fragment thereof, or a CAR-expressing immune cell (e.g. T cell, NK cell).

[0335] A method according to the present disclosure may be performed concurrently or subsequently to a CML diagnosis. That is, a method according to the present disclosure may be performed at the same time a subject is diagnosed with CML or following a subject’s CML diagnosis. A method according to the present disclosure may be performed within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks following a subject’s CML diagnosis. A method according to the present disclosure may be performed on a sample obtained from a subject before, at the same time as, or after a CML diagnosis. A method according to the present disclosure may be performed on a sample obtained from a subject within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks following a subject’s CML diagnosis. A method according to the present disclosure may be performed on a sample obtained from a subject who has CML or has been diagnosed with CML and has not received treatment for CML.

[0336] Accordingly, in some embodiments, the method for predicting whether a subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase is performed concurrently or subsequently to a CML diagnosis.

[0337] Accordingly, in some embodiments, the method of selecting a subject for treatment is performed concurrently or subsequently to a CML diagnosis.

[0338] As used herein, a “CML diagnosis” refers to the point at which it is determined that a subject has CML. Subject

[0339] The subject in accordance with aspects of the present disclosure may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient (e.g. a CML patient). A subject may have been diagnosed with a disease or condition requiring treatment (e.g. CML), may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[0340] In embodiments according to the present disclosure the subject is preferably a human subject. In some embodiments, the subject has CML or has been diagnosed with CML. In some embodiments, the methods of the present disclosure are performed concurrently or subsequently to a CML diagnosis. In some embodiments, the subject has been newly diagnosed with CML (e.g. in the preceding 1 , 2, 3, 4, 5, 6, 7 or 8 weeks) or is concurrently diagnosed with CML. In some embodiments, the subject has CML or has been diagnosed with CML and has not received treatment.

[0341] Kits

[0342] Aspects of the disclosure include in vitro diagnostic / prognostic methods and in vitro kits for performing such methods. In some embodiments, the kit provides agents for performing a method according to the present disclosure. The kit may be used for any of the methods described herein.

[0343] The kit may be suitable for or used for detecting / determining (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs, and / or (iv) the presence or absence of inflamed lymphoid progenitor cells in a sample. The kit may be suitable for or used for detecting / determining the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in a sample. The kit may be suitable for predicting whether a subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase. The kit may be suitable for selecting a subject for treatment. The kit and components thereof may be suitable for use in flow cytometry (e.g. multicolour flow cytometry). The kit and components thereof may be suitable for use in immunohistochemistry.

[0344] A kit provided herein comprises one or more components suitable for performing the methods described herein, in whole or in part. In some embodiments, the kit comprises a plurality of antigen-binding molecules (e.g. antibodies), wherein the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38, CD45RA, CD42A, CD10, CD90, CD123, pSTATI and IFITM1. In some embodiments, the plurality of antigen-binding molecules is further capable of detecting / binding to one or more of CD71 , CD105, CD253A, CD56, NKG2A and / or NKG2C.

[0345] In some embodiments, the kit comprises a plurality of antigen-binding molecules (e.g. antibodies), wherein the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD14, CD16, CD34, CD38, CD42A, CD19, CD10, pSTATI and IFITM1. In some embodiments, the plurality of antigen-binding molecules is further capable of detecting / binding to one or more of NKG2C+ and / or CD56.

[0346] In some embodiments, the kit comprises a plurality of antigen-binding molecules (e.g. antibodies), wherein the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38, CD45RA and CD42A. In some embodiments, the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38 and CD42A.

[0347] In some embodiments, the kit comprises a plurality of antigen-binding molecules (e.g. antibodies), wherein the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD14, CD16, CD34, CD38, CD10 and CD19.

[0348] In some embodiments, the kit comprises a plurality of antigen-binding molecules (e.g. antibodies), wherein the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38, CD90, CD45RA, CD123 and pSTATI (Ser727). In some embodiments, the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD19, CD14, CD16, CD34, CD38 and pSTATI (Ser727).

[0349] In some embodiments, the kit comprises a plurality of antigen-binding molecules (e.g. antibodies), wherein the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD14, CD16, CD34, CD38, CD10, CD19 and IFITM1.

[0350] In some embodiments, the kit comprises a plurality of antigen-binding molecules (e.g. antibodies), wherein the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD14, CD19, CD34, CD71 , CD105, CD235A, CD56, CD16, NKG2A and NKG2C. In some embodiments, the plurality of antigen-binding molecules is capable of detecting / binding to one or more of CD45, CD3, CD14, CD19, CD56, CD16 and NKG2C.

[0351] The kit may be suitable for a point-of-care in vitro diagnostic test. It may be a kit for laboratory-based testing. The kit may include instructions for use, such as an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0352] Numbered statements

[0353] The following numbered paragraphs (paras) describe particular aspects and embodiments of the present invention:

[0354] 1 . A method for predicting whether a subject is at risk of developing resistance to a treatment for chronic myelogenous leukaemia (CML) and / or progressing to blast crisis phase, wherein the subject has CML or has been diagnosed with CML, the method comprising: determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject; wherein (i) an increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML, (ii) an increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML, (iii) the presence of inflamed HSPCs, and / or (iv) the presence of inflamed lymphoid progenitor cells, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0355] 2. A method according to para 1 , wherein the method further comprises: determining (v) the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject, wherein a reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion in chronic phase CML patients who subsequently respond to treatment for CML, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

[0356] 3. The method according to para 1 or para 2, wherein a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:

[0357] (i) a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3%,

[0358] (ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs equal to or greater than 5%,

[0359] (iii) an increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently respond to treatment for CML,

[0360] (iv) an increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0361] (v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 51 %.

[0362] 4. The method according to any of paras 1 to 3, wherein a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:

[0363] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently progress to blast crisis phase; (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;

[0364] (iii) a similar or increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,

[0365] (iv) a similar or increased number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0366] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0367] 5. The method according to any one of paras 1 to 4, wherein the treatment for CML is a tyrosine kinase inhibitor.

[0368] 6. A method of selecting a subject for treatment, wherein the subject has chronic myelogenous leukaemia (CML) or has been diagnosed with CML, the method comprising:

[0369] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;

[0370] (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) is increased relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is increased relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML, (iii) inflamed HSPCs are present, and (iv) inflamed lymphoid progenitor cells are present; wherein the treatment comprises one or more of a second-generation TKI, a third-generation TKI, a STAMP inhibitor, a combination of a TKI and chemotherapy, a combination of a TKI and one or more steroids, or a stem cell transplant.

[0371] 7. The method according to para 6, wherein the method further comprises:

[0372] (c) administering the treatment to a subject selected for treatment in step (b).

[0373] 8. The method according to para 6 or para 7, wherein step (a) further comprises determining the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject; and wherein step (b) further comprises selecting the subject for treatment if the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells is reduced relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML.

[0374] 9. The method according to any one of paras 6 to 8, wherein a sample obtained from a subject selected for treatment comprises one or more of:

[0375] (i) a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3%,

[0376] (ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs equal to or greater than 5%,

[0377] (iii) an increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently respond to treatment for CML,

[0378] (iv) an increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0379] (v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 51 %.

[0380] 10. The method according to any one of paras 6 to 9, wherein a sample obtained from a subject selected for treatment comprises one or more of:

[0381] (i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently progress to blast crisis phase;

[0382] (ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;

[0383] (iii) a similar or increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,

[0384] (iv) a similar or increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0385] (v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0386] 11 . A method of selecting a subject for treatment, wherein the subject has chronic myelogenous leukaemia (CML) or has been diagnosed with CML, the method comprising:

[0387] (a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs), (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject; (b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) is reduced relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently progress to blast crisis phase, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is reduced relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase, (iii) inflamed HSPCs are not present, and (iv) inflamed lymphoid progenitor cells are not present; wherein the treatment comprises one or more of a first-generation TKI or a second-generation TKI.

[0388] 12. The method according to para 11 , wherein the method further comprises:

[0389] (c) administering the treatment to a subject selected for treatment in step (b).

[0390] 13. The method according to para 11 or para 12, wherein step (a) further comprises determining the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject; and wherein step (b) further comprises selecting the subject for treatment if the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells is increased relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

[0391] 14. The method according to any one of paras 11 to 13, wherein a sample obtained from a subject selected for treatment comprises one or more of:

[0392] (i) a proportion of megakaryocytic progenitor cells among HSPCs less than or equal to 3%,

[0393] (ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs less than or equal to than 5%,

[0394] (iii) a reduced number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently progress to blast crisis phase,

[0395] (iv) a reduced number / proportion of lymphoid progenitor cells that arpe IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and

[0396] (v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells equal to or greater than 51 %.

[0397] 15. The method according to any one of paras 11 to 14, wherein a sample obtained from a subject selected for treatment comprises one or more of:

[0398] (i) a similar or reduced number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients who subsequently respond to treatment for CML; (ii) a similar or reduced number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML;

[0399] (iii) a similar or reduced number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently respond to treatment for CML,

[0400] (iv) a similar or reduced number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and

[0401] (v) a similar or increased number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML.

[0402] 16. A method according to any one of paras 1 to 15, wherein:

[0403] (i) determining the number / proportion of megakaryocytic progenitor cells comprises determining the number / proportion of CD42A+ cells among HSPCs; and / or

[0404] (ii) determining the number / proportion of lymphoid progenitor cells comprises determining the number / proportion of CD19+ and / or CD10+ cells among CD34+ HSPCs; and / or

[0405] (v) determining the number / proportion of adaptive NK cells comprises determining the number / proportion of NKG2C+ cells among CD56dimCD16bri9htNK cells.

[0406] 17. A method according to any one of paras 1 to 16, wherein:

[0407] (i) determining the number / proportion of megakaryocytic progenitor cells comprises determining the number / proportion of CD42A+ cells among lin- CD45+ CD34+ cells; and / or

[0408] (ii) determining the number / proportion of lymphoid progenitor cells comprises determining the number / proportion of CD19+ CD10+ cells among lin- CD45+ CD34+ CD38+ cells; and / or

[0409] (iii) determining the number / proportion of lin- CD45+ CD34+ cells that are pSTATI -positive; and / or

[0410] (iv) determining the number / proportion of lin- CD45+ CD34+ CD42A+ cells that are IFITMI- positive; and / or

[0411] (v) determining the number / proportion of adaptive NK cells comprises determining the number / proportion of NKG2C+ cells among lin- CD45+ CD56dimCD16bri9htNK cells.

[0412] 18. A method according to any one of paras 1 to 17, wherein the patient sample has been obtained from peripheral blood or bone marrow.

[0413] 19. A kit suitable for performing the method according to any one of paras 1 to 18.

[0414] 20. The kit according to para 19, wherein the kit comprises a plurality of antigen-binding molecules, wherein the plurality of antigen-binding molecules is capable of detecting one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38, CD45RA, CD42A, CD10, CD90, CD123, pSTATI and IFITM1 , and optionally one or more of CD71 , CD105, CD253A, CD56, NKG2A and / or NKG2C. ***

[0415] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0416] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0417] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0418] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0419] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a’, ‘an’, and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about’, it will be understood that the particular value forms another embodiment.

[0420] Methods described herein may preferably be performed in vitro. The term ‘in vitro’ is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with / on intact multi-cellular organisms.

[0421] Values may be expressed herein as ‘about’ a particular value. Similarly, ranges may be expressed herein as from ‘about’ a particular value, and / or to ‘about’ another particular value. The term ‘about’ in relation to a numerical value is optional, and means for example + / - 10 %. By way of illustration, reference e.g. to ‘about 10 %’ is to be construed as 9 % to 11 %. In instances herein where ‘about’ is recited, the value it precedes is also specifically contemplated. By way of illustration, reference e.g. to ‘about 10 %’ also specifically contemplates 10 %.

[0422] Brief Description of the Figures

[0423] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[0424] Figure 1. Single cell transcriptomic-based design of flow cytometric panels to detect high- risk CP patients. (1 A) Study Design: Development of a flow cytometry-based clinical test to categorise CML patients at the time of diagnosis into 2 prognostic categories: patients with optimal European LeukemiaNet

[0019] TKI responses (CPResp), and those who eventually transformed to BC (CPBC ). The flow cytometry test comprises three main features of TKI resistance: cell lineage skewing, inflammation and adaptive NK cell accumulation defect. (1B) Graph showing bulk CD34+ CP-BC transcriptomics. Derivation of a common blast crisis signature (CBS). Genes which were commonly upregulated in CD34+ myeloid (MBC) and lymphoid (LBC) samples compared to CD34+ CP samples (>2.5 Iog2 fold change, p- adjust<0.05) were derived from a previously published microarray-based dataset [3], and used to define the CBS. (1 C) Identification of cell clusters enriched for the CBS within the single cell CML Resistance Atlas. GSEA for the CBS was performed comparing different cell clusters from CPBC and CPResp samples, and the normalized enrichment scores (NES) plotted as a heatmap. Comparisons with FDR q- value <0.05 were considered significantly enriched. Abbreviations: HSPC, CD34+ Hematopoietic stem and progenitor cells; LyP, lymphoid progenitor; MKP, megakaryocyte progenitor; iMKP, immature megakaryocyte progenitor; EOBM, eosinophil-basophil mast cell progenitor; iNeP, immature neutrophil progenitor; Pro-B cells, Pro-B; ERP, erythroid progenitor; HSC, hematopoietic stem cell; NeP, neutrophil progenitor. (1D) CBS enrichment plots for HSPCs, LyPs, and MKPs from CPBC and CPResp samples from (C). (1 E) Lineage individual CD34+ cell clusters were subjected to marker gene analysis using the Seurat pipeline, and cell surface markers used as lineage read-outs in our MFC panels. The bubble plot shows expression (color scale) while the size of the bubble represents the percent positivity of the indicated genes within individual cell clusters. (1F) (Left) Inflammation module scores for a hallmark IFNy gene set was computed for pseudobulked aggregated CD34+ HSPCs for CPBC and CPResp samples from CML Resistance Atlas. Module score p-values were determined by the Kruskal Wallis test (p<0.05). (Right) Box plot shows the expression of the IFITM1 gene within LyPs from CPBC and CPResp samples. Following Deseq-2 analysis, statistical significance was computed using the Wald test (p<0.01).

[0425] Figure 2. Inflammation within HSPCs is a powerful predictor of BC transformation at the time of CP diagnosis. (2A, 2B, 2C) Bone marrow (BM) (lighter grey symbols) or peripheral blood (PB) (darker grey symbols) mononuclear cells (MNCs) from a cohort of 28 CP CML patients who were either optimal TKI responders (CPResp) or who underwent MBC (CPMBC ) or LBC (CPLBC ) transformation were subjected to flow cytometry using the depicted antibody panels. Dotted line depicts the Youden’s J index cut-off values calculated for each panel. (2A) Flow cytometry panel for detection of lin- CD34+ CD42A+ MKPs (Panel 1) and lin- CD34+ CD38+ CD19+ CD10+ LyPs (Panel 2). Box plots show the percentages of the indicated populations. (2B) Flow cytometry panels for detection of inflammation within HSPCs (Panel 3) or LyPs (Panel 4). Left: Mean fluorescence intensity (M.F.I) of pSTATI (Ser-727) within lin- CD34+ population. Right: M.F.I of IFITM1 within the LyP identified as lin- CD34+ CD38+ CD19+ CD10+. Note: For M.F.I calculations, only samples with LyPs > 20 cells were included. Since all CPResp samples (n=8) had less than 20 LyPs, these data could not be plotted, and LyPs from normal BM (normal) was plotted as a comparison. (2C) Flow cytometry panel (Panel 5) for detection of adaptive NK cells. NKG2C+adaptive NK cell abundance within CD56dimCD16br'9htNK cells were plotted. Two-tailed Student’s t-test was used for all comparisons. (2D, 2E, 2F) Kaplan-Meier curves were plotted using positive or negative calls as determined by the Youden’s J index, and based on lineage Panels 1 and 2 (2D), inflammation Panels 3 and 4 (2E), or the adaptive NK Panel 5 (2F). The y-axis indicates the probability of BC transformation, while the x-axis is the time taken for BC transformation. Kaplan-Meier curves were compared using the Log-rank (Mantel-Cox) test. (Top) All samples including BM and PB were used for the plots. Note: When both BM and PB samples were available (n=7), only the BM sample was chosen for the top plots. p<0.0001 for (D) and (E) and p=0.03 for (F). (Bottom) Only PB samples were used to generate Kaplan-Meier curves. p<0.0072 for (D), p<0.0001 for (E) and p=0.0130 for (F). (2G) A flow cytometry-based clinical test that can quantify inflammation within HSPC lineages emerges as a powerful predictor of BC transformation at the time of CP diagnosis.

[0426] Figure 3. Receiver operating characteristic (ROC) curves were generated for the five MFC panels. The AUC (area under the curve), Youden’s J index, and cut-off points for optimal sensitivity and specificity are shown in the insets (red font).

[0427] Examples

[0428] Example 1 : Methods and materials

[0429] Sample Preparation for Flow Cytometry

[0430] CML primary patient samples were obtained from the TIDEL-II cohort (n=23) at SAHMRI, Australia, and from Singapore (n=5) at Singapore General Hospital (SGH)

[0011] . All patient samples were collected in accordance with institutional review board (IRB) protocols and with informed consent from the patients. Diagnostic bone marrow (BM) or peripheral blood (PB) samples were collected using standard protocols. Mononuclear cells (MNCs) were prepared by density gradient centrifugation using Ficoll-Paque (Sigma, MO, USA) and cryopreserved in liquid nitrogen. Either BM-MNCs or PBMNCs were used for this study based on sample availability. For 7 / 28 samples, paired BM / PB were available and both samples were used for flow cytometry. Normal healthy bone marrow (NBM) MNCs used as controls in this study were purchased from commercial sources (catalog #70001 .3, STEMCELL Technologies Inc, Canada).

[0431] Staining for flow cytometry analysis

[0432] Archived BM-MNCs or PBMCs were thawed rapidly at 37°C in complete RPMI medium containing 10% FBS. Cells were washed once and allowed to rest for 30 minutes at room temperature in the same medium before staining for flow cytometry analysis. About 200,000 MNCs were used per flow cytometry panel. For panels requiring surface staining, cells were washed with staining buffer (1% FBS-PBS + 2 mM EDTA) and incubated with the surface antibody mixture for 30 minutes on ice. For intracellular staining with anti-pSTAT1 (Ser-727) antibody, cells were washed twice with staining buffer after surface staining, fixed with 100 pL Cytofix Fixation Buffer (BD, #554714) for 15 minutes at room temperature, and washed with 1x Perm / Wash Buffer (BD, #554714). Cells were then stained with PE-pSTAT1 (Ser-727) (1 :100) for 1 hour on ice in 1x Perm / Wash Buffer (BD, #554714). Finally, cells were washed with 1x Perm / Wash Buffer (BD, #554714) before flow cytometric acquisition. For intracellular staining with IFITM1 antibody, cells were washed twice with staining buffer after the surface staining step, fixed with 100 pL Cytofix Fixation Buffer (BD, #554714) for 15 minutes at room temperature, and permeabilized by re-suspending cells in 0.1% Triton X-100 (Sigma) in PBS. Cells were then incubated with the intracellular antibody FITC- IFITM1 (1 :50) for 30 minutes at room temperature, washed twice with 0.1 % Triton X-100 PBS, and acquired on a flow cytometer (BD Fortessa). For fluorescence conjugation of IFITM1 , the FlexAble Antibody Labeling Kit (Proteintech, #KFA008) reagents following the manufacturer’s instructions. For live cell discrimination, Live / dead fixable violet dead cell stain (ThermoFisher) was used for panels 1 , 2 and 4 while Sytox Blue (ThermoFisher) was used for panel 5. Additionally, forward versus side scatter (FSC vs SSC) gating was used to identify cells of interest. All data analysis were conducted using the FlowJo™ v10 software.

[0433] Differential gene expression analysis

[0434] A pseudobulk differential gene expression re-analysis of the CML Resistance Atlas [1] using the DESeq2 pipeline [2] was performed here. Briefly, we first aggregated single-cell RNA-seq UMI counts for HSPC cell types using the AggregateExpression function from Seurat [3], resulting in a matrix where rows represented genes and columns represented patients. After filtering out genes with low expression levels, DESeq2 was employed to identify differentially expressed genes between group CPBC (Grp C from ref 1) and CPResp groups (Grp A from ref 1). Genes with statistically significant differences in pair-wise comparison (p-adjust values (<0.1) were shortlisted.

[0435] Statistical Analyses

[0436] Student’s t-test was used to for pair-wise comparison across groups. Receiver operating characteristic (ROC) analysis was performed to evaluate the performance of the flow cytometry panels and the Youden’s J index values were used to determine the optimum cut-off values [4]. The J index was calculated as "sensitivity - (1 - specificity)." Kaplan-Meier curves were compared using the Log-rank (Mantel-Cox) test. All hypotheses were constructed as two-tailed and a p-value less than 0.05 was considered statistically significant.

[0437] Example 2: Designing panels for predictive biomarker testing

[0438] The CML Resistance Atlas was interrogated to determine which sub-clusters within the hematopoietic stem and progenitor cell (HSPC) compartment of CPBC patients harboured the BC-like transcriptome we had previously identified [3]. First, by comparing bulk CD34+ chronic phase (CP) and blast crisis (BC) transcriptomes [3], a novel gene set was derived comprising 44 of the most differentially upregulated genes in both myeloid and lymphoid BC HSPCs compared to CP HSPCs, termed the ‘common BC signature’ (CBS) (Figure 1 B). Pseudobulked HSPCs from CPBC samples within the Resistance Atlas exhibited significant enrichment of the CBS compared to CPResp samples (Figure 1C).

[0439] Next, focussing on individual populations within the HSPC compartment of CPBC samples, megakaryocytic (MKP) and lymphoid (LyP) progenitor clusters were identified as being enriched for the CBS (Figure 1C and 1D). These findings prompted markers for MKP and LyP populations to be included as indicators of prognostic sub-populations expanding in TKI-resistant CML. Using inter-cluster marker gene analysis, GP9 (CD42A) was identified as an MKP marker, and MME (CD10) and CD19 as LyP markers (Figure 1 E), and these were incorporated into panels designed to detect MKP (Panel 1) and LyP (Panel 2) expansion.

[0440] For inflammation panels, it was previously noted that CPBC HSPCs display a resistance-conferring inflammatory state characterized by activation of the IFNy pathway and its downstream effector STAT1 in CPBC patients who transform to either MBC or LBC (Figure 1 F, left) [4,7]. Because STAT1 phosphorylation at serine-727 is a readout for STAT1 activity, an antibody was included directed against pSTATI (Ser-727) within the HSPC population (Panel 3) [8]. To identify additional markers of inflammation, differential gene expression analysis was conducted on HSPC clusters that harbour the CBS. IFITM1 (interferon-induced transmembrane protein 1 or CD225), an IFNy-induced inflammatory marker, [9] was found to be specifically upregulated within LyP cells, and created a LBC-specific inflammation panel (Panel 4) (Figure 1 F, right).

[0441] Lastly, a flow cytometry panel was designed as a read-out for adaptive NK cell abundance, specifically incorporating the activating NK cell receptor, NKG2C (Panel 5)

[0010] .

[0442] Example 3: Validating panels for predictive biomarker testing

[0443] The ability of the multi-colour flow cytometry-based (MFC) panels was assessed, individually or in combination, to segregate CPBC from CPResp in an independent cohort of 28 CP patients from the TIDEL II study

[0011] (n=23) and Singapore (n=5). Optimal cut-off points maximizing specificity and sensitivity for each biomarker panel were determined by calculating the Youden’s J index along the ROC curve

[0012] which was then used to classify patients as being positive or negative for each panel.

[0444] The Panels used in the experiments are described in Table 1. Using Panel 1 , which measures CD42A+ MKPs, 75% (9 / 12) of CPBC patients (CPMB Cp=0.04, CPLB Cp=0.01 ;) were correctly classified, with no false positives among CPResp patients (Figure 2A, left). With Panel 2, CD10+CD19+LyP expansion was detected in 6 / 7 (85.7%) CPLBC patients, with no false positives among CPResp or CPMBC patients (Figure 2A, right). Together, Panels 1 and 2 demonstrate aberrant lineage expansion as a sensitive (78.6%, 11 / 14) and specific (0% false positive) marker of CPBC, but with four outlier samples, S15, S16, S25 and S28, which could not be identified based on lineage Panels 1 and 2 alone.

[0445] Next, the ability of Panels 3 and 4 to detect progenitor inflammation among CPBC patients, and potentially, improve on lineage expansion-based identification was evaluated. Using Panel 3, which assesses pSTATI (Ser-727), increased HSPC inflammation in all CPMBC patients (7 / 7) and the majority of CPLBC patients (5 / 7) was observed, but none among 14 CPResp samples (p<0.01 for CPMBC; p=0.07 for CPLBC; Figure 2B, left). Using Panel 4, which detects LyP-specific inflammation through IFITM1 , it was found that all CPLBC (7 / 7) samples were positive while no healthy controls were positive (p<0.01 ; Figure 2B, right). Together, Panels 3 and 4 detected 100% (14 / 14) of CPBC patients with no false positives among CPResp patients, and importantly correctly predicted the outlier samples from Panels 1 and 2, S15, S16, S25 and S28 as being destined for BC.

[0446] Lastly, using Adaptive NK Panel 5, it was found that NKG2C+ adaptive NK cells were significantly higher in CPResp compared to CPBC patients (Figure 2C) (p<0.05).

[0447] Table 1. Backbone antibodies, markers and populations assayed in Panels 1-5

[0448] Next, the MFC data were binarized, based on marker positivity cut-offs as positive or negative for the three Hallmark features, and Kaplan-Meier curves were generated to assess the performance of the five MFC panels (Figure 2D). When CP patients were segregated according to Lineage Panels 1 and 2, the specificity and sensitivity for predicting BC transformation was 100% and 90%, respectively (p<0.0001 by log-rank test) (Figures 2D, top). Strikingly, when using Inflammation Panels 3 and 4, the specificity and sensitivity were both 100% with 92.8% of positive patients at the time of diagnosis likely to transform within 5 years (p<0.0001 by log-rank test) (Figure 2E, top). While segregation of diagnostic samples based on NK Panel 5 had a specificity of 100%, sensitivity was only 50% (p<0.03 by log-rank test) (Figure 2F, top). Consistent results were obtained when flow panel performances were evaluated using only diagnostic PB MNCs (Figure 2D, Figure 2E, Figure 2F, bottom), with 90% of inflammation-positive patients at the time of diagnosis likely to transform within 5 years (p<0.0001 by log-rank test) (Figure 2E, bottom).

[0449] In summary, these results are clinically important because they demonstrate the ability, using standard MFC, to identify patients at high-risk of BC transformation at diagnosis with high specificity and sensitivity (Figure 2G). The sensitivity and specificity values for the MFC panels detecting at-risk lineage, inflammation, or adaptive NK cell features are tabulated (using either BM or PB data for each sample based on availability.

[0450] Based on the results, any of Panels 1 , 2, 3, or 4 may be used to identify patients at high-risk of BC transformation at diagnosis. The results also show that a panel of a primary 11 -antibody tube of CD45, CD3, CD14, CD16, CD34, CD38, CD42A, CD19, CD10, p-STAT1 , and IFITM1 (integrating Panels 1-4) may be used for assessing inflammation within the HSPCs and LyP compartments (Figure 2G).

[0451] Example 4: Identification of patients at high risk of clinical resistance and / or progression to blast crisis when treated with second or third generation tyrosine kinase inhibitors

[0452] Current work has demonstrated the ability of predictive multi-color flow cytometry (pMFC) panels to identify chronic phase (CP) CML patients treated with imatinib who are at high-risk of future blast crisis (BC) transformation with high specificity and sensitivity. These pMFC tests detect aberrant inflammation as well as CML progenitor cell expansion in diagnostic CML peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNC). Such features represent established biological hallmarks that are enriched in patient who progress to BC CML. It is hypothesized that pMFC panels can also identify patients at high risk of clinical resistance and / or progression to BC when treated with second generation / third generation (2G / 3G) TKIs (tyrosine kinase inhibitors) or STAMPis (STAMP inhibitors).

[0453] This hypothesis will be tested by determining if:

[0454] 1 . In newly diagnosed CML patients, pMFC panels at enrolment and during therapy can identify patients on STAMPis who: a. Experience sub-optimal responses by ELN / NCCN criteria. b. Progress to BC.

[0455] 2. In 2L, T315I+, and Ph+ ALL, pMFC panels at enrolment and during therapy can identify patients on STAMPis who: a. Experience sub-optimal responses by ELN / NCCN criteria. b. Progress to BC.

[0456] 3. In 3L, pMFC panels at enrolment and during therapy can identify patients on STAMPis who: a. Experience sub-optimal responses by ELN / NCCN criteria. b. Progress to BC.

[0457] 4. In CP CML patients treated with first, second or third line (1 L / 2L / 3L) TKIs, pMFC panels at enrolment and during therapy can identify patients who: a. Experience sub-optimal responses by ELN / NCCN criteria. b. Progress to BC.

[0458] It is anticipated that identifying responders and non-responders at the time of CML diagnosis will enhance longer-term therapeutic outcomes and optimize patient care.

[0459] The ability to identify CP CML patients at high-risk of developing drug resistance and BC progression will help advance personalized medicine, clinical trial success and optimize resource allocation. By incorporating biomarker testing into standard practice, improved drug efficacy, reduced adverse effects, and better overall patient outcomes can be achieved. Within clinical trials, predictive biomarkers can increase the response rates of drugs by excluding patients who are destined to develop drug resistance. Such patients, particular patients who are at high risk of developing BC may benefit from more aggressive therapy, including stem cell transplants (SCT). Notably, SCTs when carried out in early-stage CP CML are associated with an approximate threefold increase in long-term survival when compared to patients transplanted in late-stage CML (1). In addition to directly benefitting patients, the use of accurate biomarkers may lead to more robust conclusions on drug efficacy, clearer results, and faster regulatory approvals. Methodology:

[0460] Patient selection:

[0461] 1 . Studies enrolling newly-diagnosed CML patients. For these studies, all patients who have progressed to BC or who have had sub-optimal clinical responses as defined by ELN and / or NCCN guidelines will be included. Samples from patients who have optimal ELN / NCCN responses will also be included as negative controls. Six control samples (i.e. patients with optimal responses) will be used for every sample from a patient who develops BC or who experiences a suboptimal clinical response. If available, follow-up samples from the same patients at week 24 and 72 will also be subjected to pMFC.

[0462] 2. Second line (2L) and third line (3L) studies. For these studies, all patients who have progressed to BC or who have had sub-optimal clinical responses as defined by ELN and / or NCCN guidelines will be included. Six control samples will be used for every sample from a patient who develops BC or who experiences a suboptimal clinical response. If available, follow-up samples from the same patients will also be subjected to MFC.

[0463] 3. For TKI studies, any CP patients on first line / second line / third line (1 L / 2L / 3L) who progressed to BC or who have had sub-optimal clinical responses as defined by ELN and / or NCCN guidelines, together with 6 control samples (i.e. patients with optimal responses).

[0464] 4. Exclusion criteria: a. Patients who switched to 2L / 3L TKIs because of drug intolerance.

[0465] Flow cytometry: pMFC will be performed on PB and BM MNCs obtained from patients enrolled on Novartis clinical trials. MNCs will be rapidly thawed at 37°C in complete RPMI medium containing 10% FBS, and washed once and before staining for flow cytometry analysis. About 200,000 MNCs will be used per flow cytometry panel. For panels requiring surface staining, cells will be washed with staining buffer (1% FBS-PBS + 2 mM EDTA) and incubated with the surface antibody mixture (see Table 1 pMFC antibody panel) for 30 minutes on ice. For intracellular staining with anti-pSTAT1 (Ser-727) antibody, cells will be washed twice with staining buffer after surface staining, fixed with 100 pL Cytofix Fixation Buffer (BD, #554714) for 15 minutes at room temperature, and washed with 1x Perm / Wash Buffer (BD, #554714). Cells will then be stained with PE-pSTAT1 (Ser-727) (1 :100) for 1 hour on ice in 1x Perm / Wash Buffer (BD, #554714), and washed with 1x Perm / Wash Buffer (BD, #554714) before flow cytometric acquisition. For intracellular staining with IFITM1 antibody, cells will be washed twice with staining buffer after the surface staining step, fixed with 100 pL Cytofix Fixation Buffer (BD, #554714) for 15 minutes at room temperature, and permeabilized by re-suspending cells in 0.1% Triton X-100 (Sigma) in PBS. Cells will then be incubated with the intracellular antibody FITC-IFITM1 (1 :50) for 30 minutes at room temperature, washed twice with 0.1% Triton X-100 PBS, and acquired on a flow cytometer (BD Fortessa). For fluorescence conjugation of IFITM1 , the FlexAble Antibody Labeling Kit (Proteintech, #KFA008) reagents following the manufacturer’s instructions. If cell numbers permit, the panel of exploratory antibodies shown in Table 2 will also be tested on the same samples in parallel.

[0466] Table 1

[0467] Table 2

[0468] Statistical analysis:

[0469] The Student’s t-test will be used for pair-wise comparison across groups. Receiver operating characteristic analysis will be performed to evaluate the performance of the flow cytometry panels and Youden’s J index values will be used to determine the optimum cut-off values [4]. The J index will be calculated as "sensitivity - (1 - specificity)." Kaplan-Meier curves will be compared using the Log-rank (Mantel-Cox) test. All hypotheses were constructed as two-tailed and a p-value less than 0.05 was considered statistically significant. scRNA- and scAT AC-seq: scRNA-seq analysis will be performed. Briefly, scATAC-seq data will be processed as follows: Nuclei will be isolated from samples using 10X single cell ATAC-seq kit instructions (10X genomics) and bar-coded single-cell libraries will be subjected to next-generation sequencing. The ArchR pipeline will be used for processing and analysing the scATAC-seq data (20). Results:

[0470] It is anticipated that samples from patients who undergo BC transformation or experience suboptimal clinical responses will have increased expression of inflammatory markers and / or expansion of CML progenitors compared to those with optimal responses. Additionally, using follow-up on-treatment samples from the same patients, the temporal changes in biomarker levels that precede clinical responses / resistance may be delineated, providing a dynamic tool for patient monitoring.

[0471] It is also possible that resistance to STAMPis may occur via mechanisms which are distinct from those that mediate TKI resistance, and that the pMFC may not work in STAM Pi-treated patients. If this this is the case, single cell sequencing technologies (scRNA-seq, scATAC-seq) may be used to discover novel mechanisms and biomarkers associated with clinical resistance to STAMPis. It is anticipated that interrogation of clinical samples using high-dimensional single cell approaches will uncover novel mechanisms of STAMPi resistance, and that this new knowledge may lead to novel biomarkers and therapeutic approaches to better predict and manage clinical STAMPi resistance.

[0472] References

[0473] 1 . Gratwohl, A., et al., Long-term outcome of patients with newly diagnosed chronic myeloid leukemia: a randomized comparison of stem cell transplantation with drug treatment. Leukemia, 2016. 30(3): p. 562-9.

[0474] 2. Brioli, A., et al., Management and outcome of patients with chronic myeloid leukemia in blast phase in the tyrosine kinase inhibitor era - analysis of the European LeukemiaNet Blast Phase Registry. Leukemia, 2024. 38(5): p. 1072-1080.

[0475] 3. Ko, T.K., et al., An integrative model of pathway convergence in genetically heterogeneous blast crisis chronic myeloid leukemia. Blood, 2020. 135(26): p. 2337-2353.

[0476] 4. Krishnan, V., et al., A single-cell atlas identifies pretreatment features of primary imatinib resistance in chronic myeloid leukemia. Blood, 2023. 141(22): p. 2738-2755.

[0477] 5. Yong, A.S., et al., High PR3 or ELA2 expression by CD34+ cells in advanced-phase chronic myeloid leukemia is associated with improved outcome following allogeneic stem cell transplantation and may improve PR1 peptide-driven graft-versus-leukemia effects. Blood, 2007. 110(2): p. 770-5.

[0478] 6. McWeeney, S.K., et al., A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic-phase chronic myeloid leukemia patients treated with imatinib. Blood, 2010. 115(2): p. 315-25.

[0479] 7. Held, S.A., et al., Interferon gamma modulates sensitivity of CML cells to tyrosine kinase inhibitors. Oncoimmunology, 2016. 5(1): p. e1065368.

[0480] 8. Varinou, L., et al., Phosphorylation of the Statl transactivation domain is required for full- fledged IFN-gamma-dependent innate immunity. Immunity, 2003. 19(6): p. 793-802.

[0481] 9. Lewin, A.R., et al., Molecular analysis of a human interferon-inducible gene family. Eur J Biochem, 1991. 199(2): p. 417-23.

[0482] 10. Lopez-Verges, S., et al., Expansion of a unique CD57(+)NKG2Chi natural killer cell subset during acute human cytomegalovirus infection. Proc Natl Acad Sci U S A, 2011 . 108(36): p.

[0483] 14725-32.

[0484] 11 . Yeung, D.T., et al., TIDEL-II: first-line use of imatinib in CML with early switch to nilotinib for failure to achieve time-dependent molecular targets. Blood, 2015. 125(6): p. 915-23. 12. Youden, W.J., Index for rating diagnostic tests. Cancer, 1950. 3(1): p. 32-5.

[0485] 13. Hehlmann, R., How I treat CML blast crisis. Blood, 2012. 120(4): p. 737-47.

[0486] 19. Hochhaus, A., et al., European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia, 2020. 34(4): p. 966-984. 20. Granja, JM., et al. ArchR is a scalable software package for integrative single-cell chromatin accessibility analysis. Nature Genetics, 2021. 53(3): p. 403-411.

Claims

1. Claims:1 . A method for predicting whether a subject is at risk of developing resistance to a treatment for chronic myelogenous leukaemia (CML) and / or progressing to blast crisis phase, wherein the subject has CML or has been diagnosed with CML, the method comprising: determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject; wherein (i) an increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently respond to treatment for CML, (ii) an increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML, (iii) the presence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and / or (iv) the presence of inflamed lymphoid progenitor cells, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

2. A method according to claim 1 , wherein the method comprises: determining each of (i), (ii), (iii), and (iv).

3. A method according to claim 1 or claim 2, wherein the method further comprises: determining (v) the number / proportion of adaptive NK cells among CD56dimCD16br'9htNK cells in the sample obtained from the subject, wherein a reduced number / proportion of adaptive NK cells among CD56dimCD16br'9htNK cells relative to the average number / proportion in chronic phase CML patients who subsequently respond to treatment for CML, indicates that the subject is at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase.

4. The method according to any one of claims 1 to 3, wherein a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:(i) a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3%,(ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs equal to or greater than 5%,(iii) an increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently respond to treatment for CML,62(iv) an increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and(v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 51 %.

5. The method according to any of claims 1 to 4, wherein a sample obtained from a subject at risk of developing resistance to a treatment for CML and / or progressing to blast crisis phase comprises one or more of:(i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently progress to blast crisis phase;(ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;(iii) a similar or increased number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive relative to the average number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,(iv) a similar or increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and(v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

6. The method according to any one of claims 1 to 5, wherein the treatment for CML comprises a tyrosine kinase inhibitor.

7. A method of selecting a subject for treatment, wherein the subject has chronic myelogenous leukaemia (CML) or has been diagnosed with CML, the method comprising:(a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;(b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells is increased relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) in chronic phase CML patients whosubsequently respond to treatment for CML, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is increased relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML,(iii) inflamed HSPCs and / or inflamed megakaryocytic progenitors are present, and (iv) inflamed lymphoid progenitor cells are present; wherein the treatment comprises one or more of a second-generation TKI, a third-generation TKI, a STAMP inhibitor, a combination of a TKI and chemotherapy, a combination of a TKI and one or more steroids, or a stem cell transplant.

8. The method according to claim 7, wherein the method further comprises:(c) administering the treatment to a subject selected for treatment in step (b).

9. The method according to claim 7 or claim 8, wherein step (a) further comprises determining the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject; and wherein step (b) further comprises selecting the subject for treatment if the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells is reduced relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML.

10. The method according to any one of claims 7 to 9, wherein a sample obtained from a subject selected for treatment comprises one or more of:(i) a proportion of megakaryocytic progenitor cells among HSPCs equal to or greater than 3%,(ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs equal to or greater than 5%,(iii) an increased number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently respond to treatment for CML,(iv) an increased number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and(v) a proportion of adaptive NK cells among CD56dimCD16bri9htNK cells less than or equal to 51 %.11 . The method according to any one of claims 7 to 10, wherein a sample obtained from a subject selected for treatment comprises one or more of:(i) a similar or increased number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently progress to blast crisis phase;(ii) a similar or increased number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase;(iii) a similar or increased number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive relative to the average number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently progress to blast crisis phase,(iv) a similar or increased number / proportion of lymphoid progenitor cells that are I FITM1 -positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and(v) a similar or reduced number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

12. A method of selecting a subject for treatment, wherein the subject has chronic myelogenous leukaemia (CML) or has been diagnosed with CML, the method comprising:(a) determining one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs, (iii) the presence or absence of inflamed HSPCs and / or inflamed megakaryocytic progenitors, and (iv) the presence or absence of inflamed lymphoid progenitor cells, in a sample obtained from the subject;(b) selecting the subject for treatment if one or more of: (i) the number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells is reduced relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently progress to blast crisis phase, (ii) the number / proportion of lymphoid progenitor cells among CD34+ HSPCs is reduced relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently progress to blast crisis phase, (iii) inflamed HSPCs and / or inflamed megakaryocytic progenitors are not present, and (iv) inflamed lymphoid progenitor cells are not present; wherein the treatment comprises one or more of a first-generation TKI or a second-generation TKI.

13. The method according to claim 12, wherein the method further comprises:(c) administering the treatment to a subject selected for treatment in step (b).

14. The method according to claim 12 or claim 13, wherein step (a) further comprises determining the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in the sample obtained from the subject; and wherein step (b) further comprises selecting the subject for treatment if the number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells is increased relative to the average number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells in chronic phase CML patients who subsequently progress to blast crisis phase.

15. The method according to any one of claims 12 to 14, wherein a sample obtained from a subject selected for treatment comprises one or more of:(i) a proportion of megakaryocytic progenitor cells among HSPCs less than or equal to 3%,(ii) a proportion of lymphoid progenitor cells among CD34+ HSPCs less than or equal to than 5%,(iii) a reduced number / proportion of HSPCs that are pSTATI -positive relative to the average number / proportion of HSPCs that are pSTATI -positive in chronic phase CML patients who subsequently progress to blast crisis phase,(iv) a reduced number / proportion of lymphoid progenitor cells that arpe IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently progress to blast crisis phase, and(v) a proportion of adaptive NK cells among CD56dimCD16bri0htNK cells equal to or greater than 51 %.

16. The method according to any one of claims 12 to 15, wherein a sample obtained from a subject selected for treatment comprises one or more of:(i) a similar or reduced number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells relative to the average number / proportion of megakaryocytic progenitor cells among hematopoietic stem and progenitor cells (HSPCs) and / or CD45+ hematopoietic cells in chronic phase CML patients who subsequently respond to treatment for CML;(ii) a similar or reduced number / proportion of lymphoid progenitor cells among CD34+ HSPCs relative to the average number / proportion of lymphoid progenitor cells among CD34+ HSPCs in chronic phase CML patients who subsequently respond to treatment for CML;(iii) a similar or reduced number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive relative to the average number / proportion of HSPCs and / or megakaryocytic progenitors that are pSTATI -positive in chronic phase CML patients who in chronic phase CML patients who subsequently respond to treatment for CML,(iv) a similar or reduced number / proportion of lymphoid progenitor cells that are IFITMI-positive relative to the average number / proportion of lymphoid progenitor cells that are IFITMI-positive in chronic phase CML patients who subsequently respond to treatment for CML, and(v) a similar or increased number / proportion of adaptive NK cells among CD56dimCD16bri9htNK cells relative to the average number / proportion of adaptive NK cells among CD56dimCD16br'9htNK cells in chronic phase CML patients who subsequently respond to treatment for CML.

17. A method according to any one of claims 1 to 16, wherein:(i) determining the number / proportion of megakaryocytic progenitor cells comprises determining the number / proportion of CD42A+ cells among HSPCs and / or CD45+ hematopoietic cells; and / or(ii) determining the number / proportion of lymphoid progenitor cells comprises determining the number / proportion of CD19+ and / or CD10+ cells among CD34+ HSPCs; and / or(v) determining the number / proportion of adaptive NK cells comprises determining the number / proportion of NKG2C+ cells among CD56dimCD16bri9htNK cells.

18. A method according to any one of claims 1 to 17, wherein:(i) determining the number / proportion of megakaryocytic progenitor cells comprises determining the number / proportion of CD42A+ cells among CD45+ lin- CD34+ cells; and / or(ii) determining the number / proportion of lymphoid progenitor cells comprises determining the number / proportion of CD19+ CD10+ cells among CD45+ lin- CD34+ CD38+ cells; and / or(iii) determining the number / proportion of inflamed HSPCs comprises determining the number / proportion of CD45+ lin- CD34+ cells that are pSTATI -positive; and / or(iv) determining the number / proportion of inflamed megakaryocytic progenitors comprises determining number / proportion of CD45+ lin- CD34+ CD42A+ cells that are pSTATI -positive;(v) determining the number / proportion of inflamed lymphoid progenitor cells comprises determining number / proportion of CD45+ lin- CD34+ cells that are IFITMI-positive; and / or(v) determining the number / proportion of inflamed lymphoid progenitor cells comprises determining number / proportion of CD45+ lin- CD34+ CD19+ CD10+ cells that are IFITMI-positive(vii) determining the number / proportion of adaptive NK cells comprises determining the number / proportion of NKG2C+ cells among CD45+ CD3- CD14- CD19- CD56dimCD16br'9htNK cells.

19. A method according to any one of claims 1 to 18, wherein the patient sample has been obtained from peripheral blood or bone marrow.

20. A kit suitable for performing the method according to any one of claims 1 to 19.

21. The kit according to claim 19, wherein the kit comprises a plurality of antigen-binding molecules, wherein the plurality of antigen-binding molecules is capable of detecting one or more of CD45, CD3, CD14, CD16, CD19, CD34, CD38, CD45RA, CD42A, CD10, CD90, CD123, pSTATI and IFITM1 , and optionally one or more of CD71 , CD105, CD253A, CD56, NKG2A and / or NKG2C.

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