Patient stratification

Abstract

The present invention relates generally to the fields of cancer therapy and patient stratification. More particularly, the specification relates to determining a likelihood of a patient response to cancer treatment.

Description

[0001] PATIENT STRATIFICATION

[0002] RELATED APPLICATION DATA

[0003] This application claims priority to United States Provisional Patent Application 63 / 741,193 filed on January 2, 2025, the contents of which are incorporated by reference in their entirety herein.

[0004] FIELD OF THE INVENTION

[0005] The specification relates generally to the fields of cancer therapy and patient stratification. More particularly, the specification relates to determining a likelihood of a patient response to cancer treatment.

[0006] BACKGROUND OF THE INVENTION

[0007] Adnexal tumours can occur on organs and organ connective tissue in almost every part of the body. Skin adnexal neoplasms comprise a wide spectrum of benign and malignant tumours that exhibit morphological differentiation towards one or more types of adnexal structures found in normal skin. Most adnexal tumours are benign, and local complete surgical excision is curative.

[0008] A malignant counterpart of almost every adnexal tumour has been described. These tumours are rare, locally aggressive, and have the potential for nodal involvement and distant metastasis, with a poor clinical outcome. Treatment for adnexal tumours depends on the specific location and types of cells involved.

[0009] The effective treatment of cancer remains an enormous challenge to clinicians, and there is a need to understand the likelihood that a treatment will be effective for any given patient, and result in clearance of cells associated with the tumours being treated.

[0010] SUMMARY OF THE INVENTION

[0011] The present inventors have identified a method of determining the likelihood of an effective patient response to cancer treatment. This has beneficial implications for patient treatment, and also for improving the quality' of clinical trials for novel cancer treatments.

[0012] Accordingly, in one aspect provided herein is a method for determining or having determined a likelihood of response to a treatment of cancer, for example basal cell carcinoma, in a subject, the method comprising detecting the presence or absence of adnexal differentiation and / or adnexal involvement in a skin lesion sample from the

[0013] 180539633.1subject, and providing a result indicating an increased likelihood of response to the treatment if no adnexal differentiation and / or adnexal involvement is detected, or providing a result indicating a decreased likelihood of response to the basal cell carcinoma treatment if adnexal differentiation and / or adnexal involvement is detected.

[0014] In some embodiments, the skin lesion has been determined to be, or likely to be, basal cell carcinoma.

[0015] In some embodiments, the method further comprises selecting the subject for a clinical trial for treatment of basal cell carcinoma if and only if no adnexal differentiation and / or adnexal involvement is detected, or excluding the subject from the clinical trial if and only if adnexal differentiation and / or adnexal involvement is detected.

[0016] In some embodiments, the method further comprises selecting the subject for the treatment if and only if no adnexal differentiation and / or adnexal involvement is detected, or excluding the subject from the treatment if and only if adnexal differentiation and / or adnexal involvement is detected.

[0017] In some embodiments, detecting the presence or absence of adnexal differentiation and / or adnexal involvement comprises staining the sample. For example, staining with haematoxylin and eosin, Periodic acid Schiff, Periodic acid Schiff and diastase, Hale’s colloidal iron stain, reticulin, and / or Prussian blue.

[0018] In a further aspect, provided herein is a method for determining or having determined a likelihood of response to a treatment of a cancer in a subject, the method comprising detecting the presence or absence of mitotic activity and / or cell proliferation in a sample from the subject, and providing a result indicating an increased likelihood of response to the treatment if mitotic activity’ and / or cell proliferation is detected, or providing a result indicating a decreased likelihood of response to the cancer treatment if mitotic activity and / or cell proliferation is not detected, wherein the treatment induces cytotoxicity through RIPK3 or procaspase 8 effector pathways.

[0019] In some embodiments, the sample is a skin lesion sample and the cancer is a cutaneous cancer.

[0020] In some embodiments, the skin lesion has been determined to be, or likely determined to be, a cutaneous cancer.

[0021] In some embodiments, the cutaneous cancer is basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, sebaceous carcinoma, primary epithelial cancers, melanoma, or primary' cancers where a subset or major subset of cells are beta-catenin positive in the nucleus, for example but not limited to colorectal, melanoma, liver, endometrial, breast, pancreas, thyroid, uterus and lung cancers.

[0022] 180539633.1In some embodiments, the cutaneous cancer is basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, sebaceous carcinoma, primary epithelial cancers, melanoma, colorectal, liver, endometrial, breast, pancreas, thyroid, uterus, or lung cancer.

[0023] In some embodiments, the method further comprises selecting the subject for a clinical trial for the treatment if and only if mitotic activity and / or cell proliferation is detected, or excluding the subject from the clinical trial if and only if mitotic activity and / or cell proliferation is not detected. For example, the subject is selected for a clinical trial if mitotic activity is detected. For example, the subject is selected for a clinical trial if cell proliferation is detected. For example, the subject is selected for a clinical trial if mitotic activity and cell proliferation is detected. For example, the subject is excluded from a clinical trial if mitotic activity is detected. For example, the subject is excluded from a clinical trial if cell proliferation is detected. For example, the subject is excluded from a clinical trial if mitotic activity and cell proliferation is detected. In some embodiments, the method further comprises selecting the subject for the treatment if and only if mitotic activity and / or cell proliferation is detected, or excluding the subject from the treatment if and only mitotic activity and / or cell proliferation is not detected. For example, the subject is selected for treatment if mitotic activity is detected. For example, the subject is selected for treatment if cell proliferation is detected. For example, the subject is selected for treatment if mitotic activity and cell proliferation is detected. For example, the subject is excluded from treatment if mitotic activity is detected. For example, the subject is excluded from treatment if cell proliferation is detected. For example, the subject is excluded from treatment if mitotic activity and cell proliferation is detected.

[0024] In some embodiments, detecting the presence or absence of mitotic activity comprises contacting the sample with a compound that binds MPM-2 or phospho-Histone H3 (PHH3), or with haematoxylin and eosin.

[0025] In some embodiments, detecting the presence or absence of cell proliferation comprises contacting the sample with a compound that binds a protein associated with cell proliferation. For example, with a compound that binds Ki-67, proliferating cell nuclear antigen (PCNA), Ki-S2, K1-S3, Ki-S5, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MCM10, CAF-1 p60, CAF-1 pl50, NAV2, Unc-53, CDC6, CDC7, CDC7 protein kinase, Dbf4, CDC14, CDC14 protein phosphatase, CDC45, topoisomerase 2 alpha, DNA polymerase delta, replication protein A (RPA), replication factor C (RFC), FEN1 or Phospho-histone H3, or any combination thereof.

[0026] 180539633.1In some embodiments, detecting the presence or absence of cell proliferation comprises determining beta-galactosidase activity. For example, presence of betagalactosidase activity indicates senescent cells, i.e. cells which are not proliferating.

[0027] In some embodiments, the compound is a binding protein. For example, the compound is an antibody.

[0028] In some embodiments, detecting the presence or absence of cell proliferation comprises a protein associated with cell proliferation. For example, determining a transcript level of Ki-67, proliferating cell nuclear antigen (PCNA), Ki-S2. Ki-S3, Ki-85, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MCM10. CAF-1 p60, CAF-1 pl50. NAV2, Unc-53, CDC6, CDC7, CDC7 protein kinase, Dbf4, CDC14, CDC14 protein phosphatase, CDC45, topoisomerase 2 alpha, DNA polymerase delta, replication protein A (RPA), replication factor C (RFC), FEN1 or Phospho-histone H3, or any combination thereof.

[0029] In some embodiments, the method further comprises administering the treatment to the subject or having the treatment administered to the subject.

[0030] In some embodiments, the treatment is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, and virotherapy. For example, the treatment consists of chemotherapy. For example, the treatment consists of radiotherapy. For example, the treatment consists of immunotherapy. For example, the treatment consists of virotherapy.

[0031] In some embodiments, the immunotherapy comprising administering a checkpoint inhibitor.

[0032] In some embodiments, the checkpoint inhibitor inhibits PD- 1 , CTLA-4. or PD-Ll, or any combination thereof.

[0033] In some embodiments, the treatment induces programmed cell death.

[0034] In some embodiments, the treatment induces cytotoxicity through RIPK3 or procaspase 8 effector pathways.

[0035] In some embodiments, the treatment comprises administering a therapeutically effective amount of a hedgehog signalling pathway inhibitor.

[0036] In some embodiments, the treatment comprises administering a therapeutically effective of a recombinant DNA virus comprising an expression cassette encoding one or more biotherapeutic agents, and operable for expression of the encoded one or more biotherapeutic agents in mammalian cancer cells.

[0037] In some embodiments, the treatment comprises administering a therapeutically effective of a recombinant DNA virus comprising an expression cassette encoding

[0038] 180539633.1interferon gamma, and operable for expression of the encoded interferon gamma in mammalian cancer cells.

[0039] Tn some embodiments, the treatment comprises administering a therapeutically effective amount of a hedgehog signalling pathway inhibitor and a recombinant DNA virus comprising an expression cassette encoding interferon gamma, and operable for expression of the encoded interferon gamma in mammalian cancer cells.

[0040] In some embodiments, the treatment comprises intralesional administration of the recombinant DNA virus.

[0041] In some embodiments, the subject to be treated is a human subject.

[0042] In some embodiments, the method further comprises obtaining the sample from the subject prior to the detection step.

[0043] In a further aspect, provided herein is a method for stratifying or having stratified a group of human subjects for a clinical trial of a candidate treatment for basal cell carcinoma, the method comprising detecting the presence or absence of adnexal differentiation and / or adnexal involvement in a skin lesion sample from a subject in the group of human subjects, and selecting the subject for the clinical trial if no adnexal differentiation and / or adnexal involvement is detected, or excluding the subject from the clinical trial if adnexal differentiation and / or adnexal involvement is detected. For example, detecting presence or absence of adnexal differentiation in a skin lesion sample from a subject. For example, detecting adnexal involvement in a skin lesion sample from a subject. For example, detecting presence or absence of adnexal differentiation and adnexal involvement in a skin lesion sample from a subject. For example, selecting the subject for the clinical trial if no adnexal differentiation is detected. For example, selecting the subject for the clinical trial if no adnexal involvement is detected. For example, selecting the subject for the clinical trial if no adnexal differentiation and no adnexal involvement is detected. For example, excluding the subject from clinical trial if adnexal differentiation is detected. For example, excluding the subject from clinical trial if adnexal involvement is detected. For example, excluding the subject from clinical trial if adnexal differentiation and adnexal involvement is detected.

[0044] In a further aspect, provided herein is a method for treating or having treated basal cell carcinoma in a subject, the method comprising administering a therapeutic agent to the subject, wherein the subject to be treated was determined to have a likelihood of a response to the therapeutic agent using a method described herein.

[0045] In a further aspect, provided herein is a use of a therapeutic agent in the manufacture of a medicament for treating basal cell carcinoma in a subject determined

[0046] 180539633.1to have a likelihood of a response to the therapeutic agent using a method described herein.

[0047] Tn a further aspect, provided herein is a therapeutic agent for use in treatment of basal cell carcinoma in a subject determined to have a likelihood of a response to the therapeutic agent using a method described herein.

[0048] In a further aspect, provided herein is a method for stratifying or having stratified a group of human subjects for a clinical trial of a candidate treatment for cancer, the method comprising detecting the presence or absence of mitotic activity and / or cell proliferation in a sample from a subject in the group of human subjects, and selecting the subject for the clinical trial if mitotic activity and / or cell proliferation is detected, or excluding the subject from the clinical trial if no mitotic activity and / or cell proliferation is not detected. For example, the subject is selected for treatment if mitotic activity is detected. For example, the subject is selected for treatment if cell proliferation is detected. For example, the subject is selected for treatment if mitotic activity and cell proliferation is detected. For example, the subject is excluded from treatment if mitotic activity is detected. For example, the subject is excluded from treatment if cell proliferation is detected. For example, the subject is excluded from treatment if mitotic activity and cell proliferation is detected.

[0049] Tn a further aspect, provided herein is a method for treating or having treated cancer in a subject, the method comprising administering a therapeutic agent to the subject, wherein the subject to be treated was determined to have a likelihood of a response to the therapeutic agent using a method described herein.

[0050] In a further aspect, provided herein is a use of a therapeutic agent in the manufacture of a medicament for treating cancer in a subject determined to have a likelihood of a response to the therapeutic agent using a method described herein.

[0051] In a further aspect, provided herein is a therapeutic agent for use in treatment of cancer in a subject determined to have a likelihood of a response to the therapeutic agent using a method described herein.

[0052] In a further aspect, provided herein is a method for excluding analysis of a response outcome in a clinical trial for treatment of basal cell carcinoma in a subject, the method comprising, following the treatment, detecting the presence or absence of adnexal differentiation and / or adnexal involvement in a skin lesion sample from the subject, and providing a result indicating inclusion of the response outcome for the analysis if no adnexal differentiation and / or adnexal involvement is detected, or providing a result indicating exclusion of the response outcome if adnexal differentiation and / or adnexal involvement is detected. For example, following treatment, presence or

[0053] 180539633.1absence of adnexal differentiation in a skin lesion sample from the subject is detected. For example, following treatment, presence or absence of adnexal involvement in a skin lesion sample from the subject is detected. For example, following treatment, presence or absence of adnexal differentiation and adnexal involvement in a skin lesion sample from the subject is detected. For example, the response outcome is included for analysis if no adnexal differentiation is detected. For example, the response outcome is included for analysis if no adnexal involvement is detected. For example, the response outcome is included for analysis if no adnexal differentiation and adnexal involvement is detected. For example, the response outcome is excluded from analysis if no adnexal differentiation is detected. For example, the response outcome is excluded from analysis if no adnexal involvement is detected. For example, the response outcome is excluded from analysis if no adnexal differentiation and adnexal involvement is detected.

[0054] The steps, features, integers, compositions and / or therapeutic agents disclosed herein or indicated in the specification of this application individually or collectively, and any combinations of two or more of said steps or features.

[0055] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0056] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0057] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (e.g. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0058] The invention is hereinafter described by way of the following non-limiting Examples.

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] General Techniques and Definitions

[0061] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, cell biology, viral vector construction, gene therapy, molecular genetics, cancer biology, cancer therapy, immunology, pharmacology, protein chemistry, and biochemistry).

[0062] 180539633.1Unless otherwise indicated, the cell culture and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach. Volumes 1 and 2, IRL Press (1991). D.M. Glover and B.D. Hames (editors). DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present). Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology', John Wiley & Sons (including all updates until present).

[0063] As used herein, the term about, unless stated to the contrary7, refers to + / - 10%, more preferably + / - 5%, of the designated value.

[0064] Throughout this specification the word '‘comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0065] As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive '‘or.” That is, unless specified otherwise, or clear from context, “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then “X employs A or B” is satisfied under any of the foregoing instances. Further, at least one of A and B and / or the like generally means A or B or both A and B. In addition, the articles “a” and “an” as used in this application and the appended claims may generally be construed to mean “one or more” unless specified otherwise or clear from context to be directed to a singular form.

[0066] As used herein, the term “immunotherapies” or “immunotherapy” means an approach for modulating the immune system of a subject, for example, to treat cancer. Examples of immunotherapies include immune checkpoint inhibitors, adoptive cell transfer (ACT) therapies including using genetically edited T-cells, and cancer vaccines, for example compositions comprising cancer antigens.

[0067] As used herein, the term '‘chemotherapy” has the meaning common in the art and includes the treatment of disease using chemical substances, including cytotoxic substances to treat cancer.

[0068] 180539633.1As used herein, the term “radiotherapy” has the meaning common in the art and includes a cancer treatment that uses high doses of radiation to kill cancer cells and shrink tumours. For example, using radiation from x-rays, gamma rays, neutrons, protons, and other sources.

[0069] As used herein, the term “virotherapy” refers to the use of therapeutic viruses to treat diseases. For example, the use of oncolytic viruses to treat cancer. Virotherapy includes use of therapeutic viruses that selectively replicate in tumours and kill cancer cells, and viruses used as a gene expression platform to induce direct cytotoxic or immune-related antitumour activity. Oncolytic viruses have evolved from early conservatively designed attenuated vectors focused on safety’ to now more complex engineered therapeutic viruses. These newer generation vectors combine enhanced selective viral replication within tumour cells using cancer conditional gene expression or entry’ with immunomodulatory gene expression to enhance the antitumor activity' of the recruited immune cells.

[0070] The term “chemotherapeutic agent” refers to a class of small molecules that is cytostatic and / or cytotoxic to cancer cells.

[0071] The terms “effective amount” or “therapeutically^ effective amount”, as used herein, refer to a sufficient amount of an administered agent (e.g., a recombinant virus, a chemotherapeutic agent, a purified protein, or a caspase inhibitor) which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. A “therapeutically effective amount” of a therapeutic agent that is administered as part of a combination treatment can refer to an amount of the therapeutic agent that would be therapeutically effective when used on its own (z.e., as a monotherapy), or may refer to a reduced amount that is therapeutically effective by virtue of its combination with one or more additional therapeutic agents.

[0072] The term “therapeutic agent,” as used herein, refers to any molecule that plays a direct role in treating a cancer. Such therapeutic agents include by way of example only, a chemotherapeutic agent, an expressed biotherapeutic agent, a recombinant virus, a purified cytokine, an antibody, a caspase inhibitor, an immune response-inducing or enhancing agent, a purified polarising cytokine, and a purified chemokine.

[0073] The terms “treating” or “treatment,” as used herein, refer to both direct treatment of a subject by a medical professional (e.g., by administering a therapeutic agent to the subject), or indirect treatment, effected, by at least one party7, (e g., a medical doctor, a nurse, a pharmacist, or a pharmaceutical sales representative) by providing instructions,

[0074] 180539633.1in any form, that (i) instruct a subject to self-treat according to a claimed method (e.g., self-administer a drug) or (ii) instruct a third party to treat a subject according to a claimed method. Also encompassed within the meaning of the term '‘treating’’ or “treatment” are prevention of relapse or reduction of the disease to be treated, e.g., by administering a therapeutic at a sufficiently early phase of disease to prevent or slow its progression. For example, the methods described herein include treatment of a cancer for example a basal cell carcinoma.

[0075] As used herein, the term “response” in the context of a response to a treatment, for example a cancer treatment, means the extent to which a patient improves, or at least partially obtains a desired therapeutic outcome, following the administration of a therapeutic intervention or treatment. A patient may experience a complete response, a partial response, stable disease / disease stabilisation, or progressive disease / disease progression. As used herein, a response can be complete or partial, for example, as determined by clinical assessment. Sometimes, a patient with clinically determined (for example, by visual assessment and / or palpation) complete response still has residual disease, as gauged or determined by pathology. Accordingly, a complete response, both clinically and pathologically determined, is the best outcome for a patient and effectively means they are disease free. However, a partial response, whether determined clinically and / or pathologically, is still atherapeutic improvement for the patient, and indicates that the patient has responded to the treatment, i.e. in a positive or beneficial way.

[0076] In some embodiments, a subject has a response to treatment if, after treatment, they have no residual disease. As used herein, “residual disease” refers to any disease present after treatment, for example as determined by clinical and / or pathological assessment. For example, a subject does not have a response to treatment if after treatment, they have residual disease. “After treatment” means at a predetermined time following a therapeutic intervention, for example upon completion of a series or course of interventions, including combination therapy, or at a time following completion of a therapeutic intervention such that the skilled person would expect the therapeutic intervention to have had its full effect.

[0077] As used herein, the term “increase” or “increases” or “increased” or “increasing” refers to having a higher or greater level of a given parameter after a specific event, for example, after receiving treatment, for example, after performing the methods described herein. For example, increased compared to the level of a given parameter at baseline, for example increased in one subject compared to another subject. For example, an increased likelihood of response to a treatment.

[0078] 180539633.1The term “subject” is any animal. In an embodiment, the subject is a mammal. In an embodiment, the subject is a human. In an embodiment, the subject is selected from a / an: neonate, infant, child, adolescent and adult. In an embodiment, the subject is a neonate. In an embodiment, the subject is an infant. In an embodiment, the subject is a child. In an embodiment, the subject is an adolescent. In an embodiment, the subject is an adult. In an embodiment, the subject has a cancer. In an embodiment the subject may or may not be aware that they have cancer, for example, the subject has been diagnosed with cancer, for example, the subject has not been diagnosed with cancer. In an embodiment, the subject has a skin cancer, for example, basal cell carcinoma. In an embodiment, the subject has a skin cancer. In an embodiment, the subject has a basal cell carcinoma.

[0079] As used herein, the term “detect”, or “detecting” means to identify, determine, or discover the existence of something, and includes in its meaning to measure, analyse, assess, and assay.

[0080] As use herein, the term “determine”, “having determined”, or “determining” means to ascertain, establish, or leam something, for example, by research or calculation. In some embodiments, the likelihood of a response to treatment is determined by the methods described herein.

[0081] As used herein, the term “sample” refers to a biological sample of any type of biological material derived from a subject that can analysed, for example, any cell, tissue, or product thereof, any biopsy, or biological fluid or fraction thereof. For example, the sample is a biopsy. For example, the sample is a tumour biopsy. For example, the sample is a skin lesion sample. In some embodiments, the methods described herein further comprise obtaining a sample from a subject prior to any detecting step.

[0082] As used herein, “skin lesion” refers to an area of skin that is different from the surrounding skin. In some embodiments, the skin lesion sample is or has been determined likely to be basal cell carcinoma. In some embodiments, the skin lesion sample is or has been determined to be cancerous.

[0083] Adnexal Differentiation and Adnexal Involvement

[0084] Adnexa is the tissue that adjoins or connects an organ and / or other associated anatomical parts, for example, adnexa may be connective tissue or other organ appendages.

[0085] Adnexa are accessory structure which are in involved in protecting and / or supporting an organ. For example, the four primary adnexal structures present in normal skin are: hair follicles, sebaceous glands (hair follicles and sebaceous glands are also

[0086] 180539633.1referred to a pilosebaceous unit), apocrine glands, and eccrine glands. Other prominent skin adnexal structures include, blood vessels, nerve fibres, sweat ducts, erector pilli muscles etc.

[0087] As used herein, the term “adnexal differentiation” refers to cellular differentiation towards an adnexal structure, i.e. in the case of skin adnexal differentiation, towards hair follicles, sebaceous glands, apocrine glands, or eccrine glands, regardless of the origin of the cell undergoing differentiation. Adnexal differentiation includes differentiation towards other adnexal structures beyond those specific to skin, for example, fallopian tubes and ovaries in the case of the uterus, for example, eyeball accessories such as the orbit, extraocular muscles, lacrimal system and optic nerves in the case of the eye, or differentiation towards any accessory structures of an organ. This variation in adnexal differentiation can make it difficult to diagnose, and neoplasm may display more than one type of adnexal differentiation. For example, Skin adnexal neoplasms (tumours) exhibit morphological differentiation towards one or more types of adnexal structures found in normal skin. For example, a basal cell carcinoma may have adnexal differentiation if it comprises cancer cells that differentiation towards an adnexal type phenotype and morphology, for example the cancer begins to adopt adnexal structure features, but those features are not a fully -formed adnexal structure.

[0088] As used herein, “adnexal involvement” refers the situation when a fully-formed adnexal structure, for example a hair follicle, a gland, muscle, vessels, tubes etc, is located in close proximity to a neoplasm, for example, next to or within a tumour.

[0089] Adnexal tumours, and therefore adnexal differentiation and / or involvement, are usually classified using histological evaluation, and classified according to the predominant morphological component. The majority of adnexal tumors can be classified into different subgroups on the basis of light microscopy alone. In some embodiments, detecting the presence or absence of adnexal differentiation comprises staining the sample. For example, staining with haematoxylin and eosin, Periodic acid Schiff, Periodic acid Schiff and diastase, Hale’s colloidal iron stain, reticulin, and / or Prussian blue.

[0090] The skilled person will be aware of the numerous publications dedicated to identification of adnexal tumours based on morphological features using haematoxylin and eosin staining, see, for example, Kazakov et al. (2013), Alsaad et al. (2007), Wheeler et al. (2023) and Givens et al. (2009).

[0091] Periodic acid Schiff (PAS) is a staining method used to detect polysaccharides, manifested as glycogen, glycoproteins, glycolipids, and mucin in tissues. PAS stains basement membrane, so can distinguish different adnexal tumours that may look similar

[0092] 180539633.1morphologically, but which are in fact comprised of different components, for example distinguishing Cylindroma (basaloid cells) from melanoma (no basaloid cells). The PAS with diastase staining procedure can be used to differentiate glycogen granules from other granules in various tumour types. Reticulin is another special stain used to exclude adnexal tumour.

[0093] Hale's colloidal iron stain for acid mucin is useful to highlight stromal mucinous degeneration in certain adnexal tumours, and Prussian blue may be useful in demonstrating iron deposits within apocrine lesions.

[0094] Beta-catenin

[0095] Beta-catenin is a critical component of cadherin-based cell-cell adhesion, has a central role in transcriptional regulation in the Wnt signalling pathway, and is an important intermediate in many other signal transduction pathways, such as the phosphoinositide 3-kinase(PI3K) / AKT pathway. Its deregulation is important in the genesis of a number of human malignancies, particularly cancers.

[0096] Beta-catenin is located at the cell membrane, and the cytoplasm and / or nucleus. At the cell membrane, it is bound to the cytoplasmic domain of type I cadherins, a complex stabilised by serine / threonine phosphorylation of beta-catenin. Other phosphorylation events, however, such as tyrosine phosphorylation of beta-catenin by epidermal growth factor receptors or Src, among others, lead to its dissociation from the adherens complex and transfer to the cytoplasm. Cytosolic beta-catenin is subsequently degraded or translocated to the nucleus. In the absence of Wnt ligands, beta-catenin binds to a complex formed by glycogen synthase kinase 30 (GSK30), adenomatous polyposis coli, and axin. This complex facilitates the phosphorylation of the NH2 terminus of 0-catenin by GSK30 or other kinases, targeting it for degradation by the ubiquitin-proteasome pathway. Activation of Wnt signaling is initiated by Wnt ligands binding to two receptor molecules, frizzled proteins and lipoprotein receptor-related proteins 5 and 6 (LRP-5 / 6). This leads to GSK30 inactivation and beta-catenin accumulation in the cytoplasm with translocation to the nucleus. There it interacts with TCF / LEF to control transcription of downstream target genes, such as cyclin DI and c-Myc. Accordingly, uncomplexed beta-catenin can be used as a proxy for activated wnt signalling, and / or as a proxy for adnexal involvement and / or adnexal differentiation. Accordingly, determining a level of uncomplexed beta-catenin can be used in the methods disclosed herein to detect the presence or absence of adnexal differentiation and / or adnexal involvement. For example, a level of beta-catenin in a sample may be compared to a reference sample. For example, a level of beta-catenin in a sample may be compared to

[0097] 180539633.1a pre-determined level. Further, determining beta-catenin cellular localisation can indicate wnt activation and / or adnexal differentiation and / or adnexal involvement.

[0098] The skilled person will be aware of suitable ways to detect and measure un complexed beta-catenin. For example, un complexed beta-catenin may be measured by capture assay using glutathione S-transferase (GST) fused to the beta-catenin binding domain of E-cadherin synthesised in E. coli. Immunoblot analysis of uncomplexed and total beta-catenin can be performed, for example using beta-catenin primary antibodies and secondary antibodies, for example, secondary antibodies conjugated to a detectable label, for example a fluorophore. Quantification of immunoreactivity can be performed by imaging, and positive and negative controls may be used. For an example of using beta-catenin to study wnt activation, see e.g. Shapiro et al. (2013), and Lopez-Knowles et al. (2010).

[0099] Cell Proliferation and / or Mitotic Activity

[0100] The cell cycle is composed of multiple phases, including the G1 phase where cells increase in size, the S phase where DNA is newly synthesised, the G2 phase where further cell growth occurs, and finally the M, or mitosis, phase where the cell ultimately divides. Some cells exit the cell cycle and stop dividing altogether; this is known as quiescence or resting state (GO). Cells decide to proliferate or remain quiescent using signalling pathways that link information about the cellular environment to the G1 phase of the cell cycle.

[0101] As used herein, the term ''mitotic activity” refers to any activity having to do with the presence of dividing cells. Mitotic activity includes measurements such as mitotic rate (number of dividing cells by mm2) and mitotic index (number of cells undergoing mitosis compared to number of cells not undergoing mitosis), but is not limited to either of these things.

[0102] As used herein, "cell proliferation” or "proliferating cells” refers to cells in the active phases of the cell cycle (Gl, S, G2 and mitosis), cells which are not quiescent, and which are not in the resting state (GO).

[0103] Cell proliferation is an increase in cell number due to cell division, or cytokinesis, the final step of the cell cycle. An actively dividing cell is proliferating, but cells can be in the proliferation cycle (active phase of the cell cycle) even when they are not actively dividing, for example, because they are preparing to divide. These cells may still be referred to in the art as proliferating cells. Cell proliferation is necessary for normal tissue development and maintenance over the lifespan. Cell proliferation is a tightly regulated

[0104] 180539633.1process, with many different proteins controlling cell cycle checkpoints. Genetic mutations found in cancer cells cause uncontrolled cellular proliferation.

[0105] Cell proliferation and mitotic activity are distinct events with significant interplay between. For example, mitogenic signals play a role in regulation of the G1 phase, and mitogenic growth factors activate G1 cyclins. For example, D-type cyclins, although not an obligate component of the cell cycle machinery, couple extracellular mitogenic signals to the Gl / S transition.

[0106] Accordingly, changes or disruptions to mitotic activity and mitogenic signals also impact cell proliferation signalling, for example, cyclin D repression and inappropriate cyclin-D-dependent CDK4 / 6 activity represents the most frequent alteration of human cyclins in cancer and bears clear pathological significance, and the skilled person will be aware, whereas a low level of G1 CDK activity is sufficient to support cell proliferation in response to normal physiological levels of mitogens, significantly higher levels of G1 CDK activity' are required to sustain hyperproliferative stimulation, such as those elicited by activated oncogenes. For a review on signalling pathways controlling cell proliferation, see, e.g. Duronio and Xiong (2013).

[0107] In some embodiments, detecting the presence or absence of mitotic activity^ comprises contacting the sample with a compound that binds MPM-2 or phospho Histone H3 (PHH3), or with haematoxylin and eosin.

[0108] In some embodiments, detecting the presence or absence of cell proliferation includes performing cell cycle assays. For example, by incorporating propidium iodide or BrdU into DNA. For example, detecting the presence or absence of cell proliferation by propidium iodide staining. For example, detecting the presence or absence of cell proliferation by BrdU assay.

[0109] Another common method for measuring cell proliferation is by examining the expression levels of cell cycle proteins and other proteins required for proliferation, of which KI is a universally accepted marker. A non-exhaustive list of common protein markers for proliferating cells are listed in Table 1.

[0110] Table 1: Some common proliferation markers

[0111]

[0112] 180539633.1

[0113]

[0114] 180539633.1

[0115]

[0116] In some embodiments, detecting the presence or absence of cell proliferation comprises contacting the sample with a compound that binds Ki-67, proliferating cell nuclear antigen (PCNA), Ki-S2. Ki-S5. MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MCM10, CAF-1 p60, CAF-1 pl50, Pomfil2, Unc-53, CDC6, CDC7, CDC7 protein kinase, Dbf4, CDC14, CDC14 protein phosphatase, CDC45, topoisomerase 2 alpha, DNA polymerase delta, replication protein A (RPA), replication factor C (RFC) or FEN1.

[0117] In some embodiments, the compound is an antibody. For example, monoclonal antibodies, bispecific antibodies, multispecific antibodies, fusion peptides, fusion diabodies, triabodies, heteroconjugate antibodies, chimeric antibodies, antibody fragments, nanobodies, and other antibody-like molecules. In some embodiments the compound is a monoclonal antibody. In some embodiments the compound is abispecific antibody. In some embodiments the compound is a multispecific antibody. In some embodiments the compound is a fusion peptide. In some embodiments the compound is a fusion diabody. In some embodiments the compound is a triabody. In some embodiments the compound is a heteroconjugate antibody. In some embodiments the compound is a chimeric antibody. In some embodiments the compound is an antibody fragment. In some embodiments the compound is a nanobody. In some embodiments the compound is an antibody-like molecule, for example a binding agent, or a binding protein.

[0118] The term "antibody" as used herein includes monoclonal antibodies, bispecific antibodies, fusion diabodies, triabodies, heteroconjugate antibodies, chimeric antibodies including intact molecules as well as fragments thereof, nanobodies and other antibody-

[0119] 180539633.1like molecules. For example, an antibody binds to the antigen(s) of the disclosure to form an antibody-antigen complex. In an embodiment, the antibody is an antibody fragment. In some embodiments, an antibody is used for detecting the presence or absence of cell proliferation. In some embodiments, an antibody is used to identify or exclude adnexal differentiation. In some embodiments, an antibody is used in therapy, for example, in methods to determine the likelihood of a patient response, for example as a co-therapy, for example an immunotherapy, for example a check point inhibitor.

[0120] Cancers

[0121] Loss of control of the cell cycle is usually a critical step in cancer development and cell-cycle aberrations are a hallmark of cancer. Cells become abnormal and processes regulating normal cell division are disrupted. Cancer cells are caught in an unregulated cell cycle. The skilled person will be aware of an abundance of literature correlating expression of the proliferation markers listed in Table 1 above with numerous and varied cancers, and the importance of regulators and actors in cell proliferation and mitosis in the progression of cancers generally, as well as the impact of the presence of cell proliferation and mitosis markers on cancer prognosis. Disrupted cell proliferation and / or mitosis is involved in all types of cancer, and the skilled person will be aware of the specific details relating to any relevant distinct type of cancer.

[0122] Without limiting the types of cancer contemplated herein, relevant cancers include: carcinomas, sarcomas, lymphomas, and melanomas. For example, skin (cutaneous), brain, breast, lung, colorectal, prostate, cervical, uterine, ovarian, and pancreatic cancer. In some embodiments, the subject has a cutaneous cancer. For example, basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, sebaceous carcinoma, or melanoma. In some embodiments, the subject has basal cell carcinoma. I some embodiments, the subject has squamous cell carcinoma. In some embodiments, the subject has Merkel cell carcinoma. In some embodiments, the subject has dermatofibrosarcoma protuberans. In some embodiments, the subject has sebaceous carcinoma. In some embodiments, the subject has melanoma.

[0123] In some embodiments, the methods described herein comprise detecting the presence or absence of mitotic activity and / or cell proliferation in a sample from the subject. For example, the presence of mitotic activity is detected. For example, the presence of cell proliferation is detected. For example, the presence of mitotic activity and cell proliferation is detected. For example, the absence of mitotic activity is detected. For example, the absence of cell proliferation is detected. For example, the absence of

[0124] 180539633.1mitotic activity and cell proliferation is detected. For example, the presence of mitotic activity and the absence of cell proliferation is detected. For example, the absence of mitotic activity and the presence of cell proliferation is detected.

[0125] In some embodiments, the methods described herein comprise providing a result indicating an increased likelihood of response to treatment, for example, cancer treatment, if mitotic activity and / or cell proliferation is detected, or providing a result indicating a decreased likelihood of response to treatment, for example cancer treatment, if mitotic activity and / or cell proliferation is not detected. For example, a subject in whose sample the presence of mitotic activity is detected is likely to have a response to treatment, i.e. for treatment to be effective. For example, a subject in whose sample the presence of mitotic activity cannot be detected is unlikely to have a response to treatment, i.e. for treatment to be effective. For example, a subject in whose sample the presence of cell proliferation is detected is likely to have a response to treatment, i.e. for treatment to be effective. For example, a subject in whose sample the presence of cell proliferation cannot be detected is unlikely to have a response to treatment, i.e. for treatment to be effective. For example, a subject in whose sample the presence of mitotic activity is detected is more likely to have a response to treatment, i.e. for treatment to be effective, than a subject in whose sample mitotic activity cannot be detected. For example, a subject in whose sample the presence of cell proliferation is detected is more likely to have a response to treatment, i.e. for treatment to be effective, than a subject in whose sample cell proliferation cannot be detected. For example, a subject in whose sample both mitotic activity and cell proliferation are detected is most likely to have a response, i.e., for treatment to be effective.

[0126] In some embodiments, the methods described herein comprise detecting the presence or absence of mitotic activity and / or cell proliferation in a sample from the subject, and providing a result indicating an increased likelihood of response to the treatment if mitotic activity and / or cell proliferation is detected, or providing a result indicating a decreased likelihood of response to the cancer treatment if mitotic activity and / or cell proliferation is not detected.

[0127] In some embodiments, the methods described herein comprise detecting the presence or absence of mitotic activity and / or cell proliferation in a sample from the subject, and providing a result indicating an increased likelihood of response to the treatment if mitotic activity7and / or cell proliferation is detected, or providing a result indicating a decreased likelihood of response to the cancer treatment if mitotic activity and / or cell proliferation is not detected.

[0128] 180539633.1In some embodiments, the methods described herein comprise detecting the presence or absence of mitotic activity and / or cell proliferation in a sample from the subject, and providing a result indicating an increased likelihood of response to the treatment if mitotic activity and / or cell proliferation is detected, or providing a result indicating a decreased likelihood of response to the cancer treatment if mitotic activity and / or cell proliferation is not detected.

[0129] Basal Cell Carcinoma (BCC)

[0130] Basal cell carcinoma (BCC) is the most common form of skin cancer and the most frequently occurring form of all cancers. There are approximately 3.6 million BCC diagnoses each year in the U.S. alone. While many factors can lead to a BCC, the main cause is sun exposure. BCCs usually appear on the face, neck, back or shoulders and are usually the least dangerous type of skin cancer. Because BCCs grow slowly, most are curable and cause minimal damage when caught and treated early. BCC most often occurs when DNA damage from exposure to ultraviolet (UV) radiation from the sun or indoor tanning triggers changes in basal cells in the outermost layer of skin (epidermis), resulting in uncontrolled growth. BCCs rarely spread beyond the original tumour site. But these lesions can grow and become disfiguring and dangerous. Untreated BCCs can become locally invasive, grow wide and deep into the skin and destroy skin, tissue and bone. There are some highly unusual, aggressive cases when BCC spreads to other parts of the body. In even rarer instances, this type of BCC can become life-threatening. The longer the wait until treatment, the more likely it is that the BCC will recur, sometimes repeatedly.

[0131] There are three types of BCC, superficial (limited to the top layers of the skin but can be wide), nodular (usually look like a rounded lump), and infiltrating (the most difficult to see, are often smaller than the eye can see, and not found until they have reached an advanced stage). BCC is diagnosed by taking a skin sample biopsy, for example of the skin lesion, which is laboratory tested.

[0132] Basal cell carcinoma (BCC) is the most common human skin malignancy. Traditional first line treatment for BCC involves surgical excision but this can be quite invasive and suboptimal for certain tumours and patients, particularly when multiple tumours are present. As the incidence of BCC continues to increase, there is a need for effective alternative treatments, particularly as our population ages.

[0133] In patients with Gorlin-Goltz’s Syndrome / Basal Cell Nevus Syndrome (BCNS), BCCs usually develop on the central face, chest, back and scalp in adolescence but may appear as early as the first year of life, with a median age of onset of 20 years. Patients

[0134] 180539633.1can present with between one and hundreds of BCCs, and as determined in a recent trial, can develop a median of 29 new, surgically eligible BCCs / year. The number of BCCs in these patients significantly diminish their quality of life because of their sheer number and the need for life-long treatment. BCNS remains difficult to treat because each BCC must be treated individually, or can be treated systemically with Hedgehog (Hh) inhibitors but long term tolerability is an issue. A well-tolerated curative local therapy for BCCs in BCNS patients represents an unmet medical need.

[0135] Current management of BCNS patients generally involves definitive treatment for high-risk tumours and the continued monitoring of lower risk tumours or their management with topical or oral options. Surgery is the primary curative treatment utilised for high-risk tumours and the excision of as many as 3-5 tumours per visit is common. Many BCNS patients undergo large numbers of surgical procedures annually which results in significant disfigurement, morbidity7and procedural fatigue.

[0136] It has also been reported that the number of sporadic BCC patients who develop more than one sporadic BCC is on the rise and these multiple BCC patients share many of the treatment and morbidity7challenges faced by BCNS patients.

[0137] A non-surgical modality that can concurrently treat multiple tumours (2-6 tumours) with a high cure rate and is well tolerated and can provide clinical benefit in non-injected tumours could be the basis of a new and substantially improved treatment paradigm for BCNS and sporadic BCC patients with multiple tumours.

[0138] In some embodiments, provided herein is a method for treating or having treated basal cell carcinoma in a subject, the method comprising administering a therapeutic agent to the subject, wherein the subject to be treated was determined to have a likelihood of a response to the therapeutic agent using a method described herein.

[0139] Patient Stratification

[0140] As used herein, "patient stratification'’ refers to the distribution of subjects (patients) into subgroups. For example, patients may be stratified according to the methods described herein based on the likelihood of a response to a treatment, for example, a cancer treatment. In some embodiments, patients are stratified based on the presence or absence of adnexal differentiation and / or adnexal involvement. In some embodiments, patients are stratified based on the presence or absence of mitotic activity and / or cell proliferation in a sample from the patent. In some embodiments, patients are stratified based on whether or not their sample is procaspase 8 positive. A sample is procaspase 8 positive if procaspase 8 is detected in more than 50% of the cells in a sample. In some embodiments, patients are stratified based on whether or not their

[0141] 180539633.1sample is 50% to 100% procaspase 8 positive. For example, the sample is at least 50%, or 60%, or 70%, or 80%, or 90%, or 95%, or 98%, or 99% or 100% procaspase 8 positive. In some embodiments, the sample is at least 50% procaspase 8 positive. In some embodiments, the sample is at least 60% procaspase 8 positive. In some embodiments, the sample is at least 70% procaspase 8 positive. In some embodiments, the sample is at least 80% procaspase 8 positive. In some embodiments, the sample is at least 90% procaspase 8 positive. In some embodiments, the sample is at least 95% procaspase 8 positive. In some embodiments, the sample is at least 98% procaspase 8 positive. In some embodiments, the sample is at least 99% procaspase 8 positive. In some embodiments, the sample is at least 100% procaspase 8 positive.

[0142] In some embodiments, patients are stratified based on whether their sample is mixed lineage kinase domain like pseudokinase (MLKL) positive. A sample is MLKL positive if procaspase 8 is detected in more than 50% of the cells in a sample. In some embodiments, patients are stratified based on whether or not their sample is 50% to 100% MLKL positive. For example, the sample is at least 50%, or 60%, or 70%, or 80%, or 90%, or 95%, or 98%, or 99% or 100% MLKL positive. In some embodiments, the sample is at least 50% MLKL positive. In some embodiments, the sample is at least 60% MLKL positive. In some embodiments, the sample is at least 70% MLKL positive. In some embodiments, the sample is at least 80% MLKL positive. In some embodiments, the sample is at least 90% MLKL positive. In some embodiments, the sample is at least 95% MLKL positive. In some embodiments, the sample is at least 98% MLKL positive. In some embodiments, the sample is at least 99% MLKL positive. In some embodiments, the sample is at least 100% MLKL positive.

[0143] In some embodiments, patients are stratified based on whether or not their sample is RIPK3 positive. A sample is RIPK3 positive if procaspase 8 is detected in more than 50% of the cells in a sample. In some embodiments, patients are stratified based on whether or not their sample is 50% to 100% RIPK3positive. For example, the sample is at least 50%, or 60%, or 70%, or 80%, or 90%, or 95%, or 98%, or 99% or 100% RIPK3 positive. In some embodiments, the sample is at least 50% RIPK3positive. In some embodiments, the sample is at least 60% RIPK3 positive. In some embodiments, the sample is at least 70% RIPK3 positive. In some embodiments, the sample is at least 80% RIPK3 positive. In some embodiments, the sample is at least 90% RIPK3 positive. In some embodiments, the sample is at least 95% RIPK3 positive. In some embodiments, the sample is at least 98% RIPK3 positive. In some embodiments, the sample is at least 99% RIPK3 positive. In some embodiments, the sample is at least 100% RIPK3 positive.

[0144] 180539633.1In some embodiments, patients are stratified based on detection of one or more of MPM-2, phospho Histone H3 (PHH3), Ki-67, proliferating cell nuclear antigen (PCNA), K1-S2, Ki-S3, Ki-S5, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MCM10, CAF-1 p60, CAF-1 pl50, Pomfil2, Unc-53, CDC6, CDC7, CDC7 protein kinase, Dbf4, CDC14, CDC14 protein phosphatase, CDC45, topoisomerase 2 alpha, DNA polymerase delta, replication protein A (RPA), replication factor C (RFC), FENL and beta-galactosidase activity. In some embodiments, patients are stratified based on a combination of one or more of the above listed features. For example, patients are stratified based on whether their sample is procaspase 8 positive and MLKL positive. For example, patients are stratified based on whether their sample is procaspase 8 positive and MLKL positive and RIPK3 positive. For example, patients are stratified based on whether their sample is procaspase 8 positive and RIPK3 positive.

[0145] In some embodiments, patients are stratified according to the methods described herein based on the likelihood of a response to a treatment, for example, a cancer treatment, wherein patients whose sample is procaspase 8 positive are likely to respond well to treatment, for example, are likely to not have any residual disease once treatment is completed. The greater the percentage procaspase 8 positive, the more likely the patient is to respond well to treatment. In some embodiments, patients whose sample is not procaspase 8 positive and is not MLKL positive are not likely to respond to treatment, for example, they are likely to have residual disease after completion of the treatment. In some embodiments, patients whose sample is not procaspase 8 positive, but is MLKL positive, and is RIPK3 positive, are likely to respond to treatment, for example, are likely to not have any residual disease after treatment is completed. The greater the percentage MLKL positive the sample is, the more likely the patient is to respond to treatment. The greater the percentage RIPK3 positive the sample is, the more likely the patient is to respond to treatment. In some embodiments, patients whose sample is not procaspase 8 positive, is MLKL positive, but is not RIPK3 positive, are not likely to respond to treatment, for example, are likely to have residual disease after treatment is completed.

[0146] In some embodiments, provided herein is a method for stratifying or having stratified a group of human subjects for a clinical trial of a candidate treatment for basal cell carcinoma, the method comprising detecting the presence or absence of adnexal differentiation and / or adnexal involvement in a skin lesion sample from a subject in the group of human subjects.

[0147] In some embodiments, provided herein is a method for stratifying or having stratified a group of human subjects for a clinical trial of a candidate treatment for cancer,

[0148] 180539633.1the method comprising detecting the presence or absence of mitotic activity and / or cell proliferation in a sample from a subject in the group of human subjects.

[0149] In some embodiments, patient stratification further comprises selecting a subject. For example, a subject is selected for treatment only if and only if mitotic activity and / or cell proliferation is detected. For example, a subject is selected for a clinical trial for treatment if and only if mitotic activity and / or cell proliferation is detected. For example, a subject is selected for treatment if and only if adnexal differentiation and / or adnexal involvement is not detected. For example, a subject is selected for a clinical trial for treatment if and only if adnexal differentiation and / or adnexal involvement is not detected.

[0150] In some embodiments, patient stratification further comprises excluding a subject. For example, a subject is excluded from treatment only if and only if mitotic activity and / or cell proliferation is not detected. For example, a subject is excluded from a clinical trial for treatment if and only if mitotic activity and / or cell proliferation is not detected. For example, a subject is excluded from treatment if and only if adnexal differentiation and / or adnexal involvement is detected. For example, a subject is excluded from clinical trial for treatment if and only if adnexal differentiation and / or adnexal involvement is detected.

[0151] Tn some embodiments, patient stratification is done manually. In some embodiments, patient stratification is performed by a computer, for example a processor, for example a system comprising a processor. In some embodiments, patient stratification is performed using a combination of manual and automated or computer-mediated steps.

[0152] In some embodiments, information used to stratify patients is collected, and / or analysed and / or stored manually, for example by a clinician. In some embodiments, the information used to stratify patients is collected and / or analysed and / or stored by computer.

[0153] In some embodiments, information used to stratify patients is obtained from images of patient samples. For example, histological images. In some embodiments, the sample is stained, for example with haematoxylin and eosin, for example with Periodic acid Schiff, for example with Periodic acid Schiff and diastase, for example w ith Hale’s colloidal iron stain, for example with reticulin, for example with Prussian blue, for example with BrdU, or example with propidium iodide, for example with immunohistochemical markers to detect binding to one or more of MPM-2, phospho Histone H3 (PHH3), Ki-67, proliferating cell nuclear antigen (PCNA), Ki-S2, Ki-S3, Ki-85, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MCM10, CAF-1 p60, CAF-1

[0154] 180539633.1pl50, Pomfd2, Unc-53, CDC6, CDC7, CDC7 protein kinase, Dbf4, CDC14, CDC14 protein phosphatase, CDC45, topoisomerase 2 alpha, DNA polymerase delta, replication protein A (RPA), replication factor C (RFC), FEN1 and beta-galactosidase activity.

[0155] In some embodiments, the stain is haematoxylin and eosin. In some embodiments, the stain is Periodic acid Schiff. In some embodiments, the stain is Periodic acid Schiff and diastase. In some embodiments, the stain is Hale’s colloidal iron stain. In some embodiments, the stain is reticulin. In some embodiments, the stain is Prussian blue.

[0156] In some embodiments, the compound binds Ki-67. In some embodiments, the compound binds proliferating cell nuclear antigen (PCNA). In some embodiments, the compound binds Ki-S2. In some embodiments, the compound binds Ki-S3. In some embodiments, the compound binds Ki-S5. In some embodiments, the compound binds MCM2. In some embodiments, the compound binds MCM3. In some embodiments, the compound binds MCM4. In some embodiments, the compound binds MCM5. In some embodiments, the compound binds MCM6. In some embodiments, the compound binds MCM7. In some embodiments, the compound binds MCM10. In some embodiments, the compound binds CAF-1 p60. In some embodiments, the compound binds CAF-1 pl50. In some embodiments, the compound binds NAV2. In some embodiments, the compound binds Unc-53. In some embodiments, the compound binds CDC6. In some embodiments, the compound binds CDC7. In some embodiments, the compound binds CDC7 protein kinase. In some embodiments, the compound binds Dbf4. In some embodiments, the compound binds CDC14. In some embodiments, the compound binds CDC14 protein phosphatase. In some embodiments, the compound binds CDC45. In some embodiments, the compound binds topoisomerase 2 alpha. In some embodiments, the compound binds DNA polymerase delta. In some embodiments, the compound binds replication protein A (RPA). In some embodiments, the compound binds replication factor C (RFC). In some embodiments, the compound binds FEN1. In some embodiments, the compound binds Phospho-histone H3.

[0157] In some embodiments, the images of the stained samples are analysed by¬ computer, for example, in an automated manner. In some embodiments, the images of the stained samples are analysed manually, for example by a clinician. In some embodiments, a combination of manual and automated analysis is performed.

[0158] It is contemplated by the inventors that the methods descried herein, or any individual steps of the methods, may be performed manually, for example by a human, or in an automated manner, for example by a processor, a computer, an algorithm, or a system comprising any combination thereof.

[0159] 180539633.1In some embodiments, provided herein are new and improved systems and methods that facilitate stratifying patients according to a likelihood of response to treatment, wherein likelihood of response to treatment is determined using methods described herein.

[0160] In some embodiments, provided herein is a method of ranking patients according to a likelihood of response, wherein the method comprises, for each of two or more patients: retrieving from a database information pertaining to one or more of: presence or absence of adnexal differentiation and / or adnexal involvement in a patient sample; presence or absence of mitotic activity; presence or absence of cell proliferation; whether or not the sample is procaspase 8 positive; whether or not the sample is MLKL positive; and whether or not the sample is RIPK3 positive. For example, ranking occurs by assigning a ranking score based on the information listed above. In some embodiments, the method further comprises outputting a ranked list of patients according to an assigned ranking score. In some embodiments, patients are stratified according to an assigned ranking score. In some embodiments patients are stratified according to presence or absence of above-listed features.

[0161] In some embodiments, stratification comprises one or more of: retrieving a histological image from a patient sample; analysing the image for one or more of: adnexal differentiation; adnexal involvement; mitotic activity; cell proliferation; procaspase 8 positivity; MLKL positivity; RIPK3 positivity; and analysing patient demographic and / or clinical information.

[0162] In some embodiments, stratification is performed by a system, for example a system comprising a computer-aided stratification module, for example, comprising a processor adapted to perform the steps of any of the methods described herein. In some embodiments, the system calculates a computer-aided stratification score for the patient as a function of the analysed feature and / or the patient demographic and / or clinical information. In some embodiments, the processor is further configured to output (e.g., to a user interface, including, for example, a personal hand-held device, a printer, or the like) a list of stratified patients, optionally ranked according to an assigned ranking score.

[0163] In some embodiments, the system further comprises a processor that executes and a memory that stores, computer-executable instructions for performing the various functions, methods, techniques, applications, etc., described herein.

[0164] In some embodiments, provided herein is a computer-readable medium on which is stored computer-executable instructions for patient stratification according to the methods described herein.

[0165] 180539633.1In some embodiments, patient stratification may occur post-hoc, after the event. For example, if, after inclusion in a clinical trial, a subject sample is tested according to the methods described herein and the sample would have been excluded from the trial and / or any treatment had the methods been performed prior to inclusion in the trial and / or treatment in accordance with the methods described herein, the stratification occurs post-hoc, after the event. For example, a subject may be included in a clinical trial but they may be excluded from the trial at any time point after initial inclusion, based on the methods described herein. For example, a subject may be included in a clinical trial but their data may be excluded from analysis at any time point after initial inclusion, based on the methods described herein. For example, a subject may be included in a clinical trial but they may be excluded from the trial and their data may be excluded from analysis at any time point after initial inclusion, based on the methods described herein. For example, a subj ect may receive treatment, including partial treatment, and subsequently be excluded from treatment based on the methods described herein. For example, subject data from a clinical trial for treatment of carcinoma may be excluded if, following inclusion in the clinical trial and / or treatment or partial treatment, the presence or absence of adnexal differentiation and / or adnexal involvement in a skin lesion sample from the subject is detected. For example, detection of the presence or absence of adnexal differentiation and / or adnexal involvement in a skin lesion sample from a subject after the subject has already participated in a clinical trial, including only partially, may provide a result indicating inclusion of the response outcome for the analysis if no adnexal differentiation and / or adnexal involvement is detected, or provide a result indicating exclusion of the response outcome if adnexal differentiation and / or adnexal involvement is detected.

[0166] Cancer Treatment

[0167] In some embodiments, the methods described herein included administering a treatment to the subject. In some embodiments, the subject has had treatment administered. In some embodiments, the methods include having the treatment administered to a subject. For example, the treatment is a cancer treatment.

[0168] The skilled person will be aware of suitable cancer treatments, and of the variables impacting selection of a suitable treatment in any given circumstance.

[0169] In some embodiments, provided herein is a method for treating or having treated cancer in a subject, the method comprising administering a therapeutic agent to the subject, wherein the subject to be treated was determined to have a likelihood of a response to the therapeutic agent using a method described herein.

[0170] 180539633.1In some embodiments, the treatment is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, and virotherapy. In some embodiments, the treatment is a primary treatment. In some embodiments the treatment is neoadjuvant therapy. In some embodiments, the treatment is a combination therapy. For example, two or more therapeutic interventions are made either concurrently or consecutively, for example in several doses, for example by the same or different administration routes. For example, treatment may comprise a combination regime of a chemotherapeutic agent and at least one other therapeutic agent.

[0171] The methods described herein include treatment of a cancer, for example a basal cell carcinoma. In some embodiments, the treatment induces cytotoxicity’. For example, by interfering with DNA synthesis and replication. In some embodiments, cytotoxic drugs, for example chemotherapy agents, induce an immunogenic ty pe of cell death in tumour cells, for example via indirect immune cell stimulation.

[0172] Cell death is known to occur through a number of distinct, though often interconnected pathyvays, including: apoptosis, necroptosis, parthanatos, and autophagic cell death, though other, uncharacterised, cell death pathways are likely to exist. Apoptosis, a form of programmed cell death, can be initiated through one of two pathways: an intrinsic pathway by which the cell kills itself as a direct response to stress, and an extrinsic pathyvay by which the cell kills itself in response to signals received from its microenvironment. The two pathyvays both activate initiator caspases, which ultimately activate executioner caspases that kill the cells by degrading proteins indiscriminately. Necroptosis is a caspase-independent form of cellular suicide that often occurs in response to extrinsic signals associated with inflammation, e.g., signalling by certain cytokines such as TNF-alpha. RIPK3 plays a pivotal role in the execution of necroptosis. Parthanatos, another form of programmed cell death, is caused by the accumulation of poly(ADP-Ribose) or “PAR’" and the nuclear translocation of apoptosisinducing factor (AIF) from mitochondria. Parthanatos is also known as poly(ADP-Ribose) polymerase 1 (PARP-l)-dependent cell death. PARP-1 mediates parthanatos upon activation in response to extreme genomic stress and synthesises PAR which causes nuclear translocation of AIF. Autophagic cell death, yet another distinct cell death pathyvay, is induced by extreme cell stress, and is characterised by cytoplasmic vacuolation (formation of autophagosomes) followed by excessive engulfment and degradation of organelles and other cytoplasmic components in lysosomes, which lead to cell death.

[0173] In some embodiments, the treatment induces programmed cell death. In some embodiments, treatment induces cell death via phagocytosis, necrosis, and / or apoptosis.

[0174] 180539633.1For example, treatment induces cell death via phagocytosis. For example, treatment induces cell death via necrosis. For example, treatment induces cell death via apoptosis.

[0175] As will be appreciated by the skilled person in the art, depending on the particular cancer type to be treated both in terms of tissue specificity (for example, melanoma) and mutational profile (for example, caspase-8 deficient cancers or RIPK3-deficient cancers) the cell death pathways engaged by the treatment methods described herein may differ. The ability of the treatment methods described herein to engage multiple cell death pathways is thus likely to increase the likelihood of successful treatment of a cancer.

[0176] In some embodiments, the treatment methods described herein induce cancer cell death through at least one cell death pathway selected from among apoptosis, necroptosis, parthanatos, and autophagic cell death.

[0177] The treatment will depend on the type, size and location of the cancer, as well as the subject’s age and health. For example, BCC is generally treated with invasive surgery to remove the tumour. Other treatments for BCC specifically include freezing the area with liquid nitrogen (cryotherapy) to kill the cancer cells, scraping off the spot, then using low-level electric current to seal the wound and kill cancer cells, creams, liquids and lotions that activate the immune system to destroy superficial BCCs (immunotherapy), photodynamic therapy, which uses a light source and cream to treat superficial BCCs, chemotherapy for skin cancers that affect the top layer of skin, applied to the skin as an ointment or cream, and radiotherapy which uses doses of radiation for certain BCCs where surgery cannot be used.

[0178] In some embodiments, the treatment comprises one or more of: cytotoxicity through the RIPK3 pathway; cytotoxicity through the Procaspase 8 effector pathway; a hedgehog signalling pathway inhibitor; a recombinant DNA virus; and a checkpoint inhibitor.

[0179] In some embodiments, the cancer is BCC and the treatment is selected from: ASN-002 (SP-002; see, e.g., Clinical Trial NCT02550678); VP-315 (YcanthiM-cantharidin; verrica.com / pipeline-vp-315 / ); interferons; TLR7 (9) agonist; STING agonists; cemiplimab; oncolytic vectors (that induce an immune response); hedgehog pathway inhibitors (for example, but not limited to vismodegib and sonidegib), NOTCH inhibitors, WNT inhibitors, 5-FU, Rapamycin, ingenol mebutate; imiquimod (TLR7 agoanist); siRNA immunotherapies (for example but not limited to TGF-beta / Cox-2 and siRNA SMO inhibitors); antibody fragment with IL-2 / TNF-a fusion; taladigeb (competitive SMO antagonist in SMO mutants); LEQ506 (SMO antagonist); silmitasertib (CX-4945, a highly selective casein kinase 2 (CK2) inhibitor which hinders GLI expression in the terminal steps of the hedgehog pathway, see NCT03897036); super

[0180] 180539633.1bioavailability (SUBA) itraconazole (see NCT02354261); nivolumab (PD-1 inhibitor); relatimab (lymphocyte activating gene 3 (LAG3) inhibitor and works as a type of checkpoint inhibitor by dampening the immunosuppressive function of regulatory T Cells); ipilimumab (see, e.g. NCT03521830); Vusolimogene oderparepvec (RP1) (a genetically modified oncolytic HSV that encodes granulocyte-monocyte colonystimulating factor (GM-CSF) and GALV-GP R, which enhances immunogenic cell death; see e.g. NCT04349436); pembrolizumab (see. e.g. NCT04799054); intratumoral vidutolimod (TLR9 agonist; see e.g. NCT04916002); or combinations thereof.

[0181] In some embodiments, the treatments are combined with each or other, and / or are combined with immunotherapy, radiation, and / or chemotherapy (see, for example, Idriss et al., 2024).

[0182] In some embodiments, the cancer is an epithelial cancer or an inherited conditions which includes epithelial cancer as a symptom, for example Basal Cell Nevus Syndrome, and the treatment is selected from FLD-103, FSD147L, PMO-Glil, BO-112, SM-020, SP-002, Vismodegib, EN002, 5 -Fluorouracil, Klisyri, cemiplimab IV, Cemiplimab, L19IL2, L19TNF, LTX-315, VP-315, STP705, AIV001, Imvamune, ASN-002, Patidegib, and / or CX-4945, (see clinical trial IDs ACTRN12624001138572, NCT06422936, CTIS2024-511801-51-00, NCT06409195, NCT06344052, ACTRN 12623001219673, NCT06150144, CTIS2023-505971 -66-00, ISRCTN10511385, NCT05929664, NCT05651828, NCT05561634, NCT05550766, NCT05329792, NCT05188729, NCT04918381, NCT04669808, NCT04679480, NCT04470726, NCT04410874. NCT04416516, NCT04362722, NCT04155190. ACTRN12619001298101, NCT03897036, NCT03208296, NCT03132636. NCT03035188, NCT02956889, ACTRN12615001017516. and NCT02550678).

[0183] In some embodiments, the treatment is combined with another therapeutic intervention, for example, surgery or radiotherapy.

[0184] RIPK3 signalling pathway

[0185] RIPK3 (receptor-interacting protein kinase 3) is a serine / threonine-protein kinase. As a key component of necrosomes, RIPK3 is an essential mediator of inflammatory' factors (such as TNFa-tumour necrosis factor a) and infection- induced necroptosis, a programmed necrosis. In addition, RIPK3 signalling is also involved in the regulation of apoptosis, cytokine / chemokine production, mitochondrial metabolism, autophagy', and cell proliferation by interacting with and / or phosphorylating the critical regulators of the corresponding signalling pathways.

[0186] 180539633.1The RIPK3 signalling pathway has been implicated in the pathogenesis of many types of cancers and exhibits both tumour-promoting and tumour-suppressive activities, indicating that RIPK3 plays diverse roles in tumour development, metastasis, and recurrence.

[0187] Similar to apoptosis, RIPK3-signalling-mediated necroptosis is inactivated in most types of cancers, suggesting RIPK3 might play a critical suppressive role in the pathogenesis of cancers. However, in some inflammatory types of cancers, such as pancreatic cancers and colorectal cancers, RIPK.3 signalling might promote cancer development by stimulating proliferation signalling in tumour cells and inducing an immunosuppressive response in the tumour environment. For a recent review of RIPK3 and its role in cancers, see e g. Liu et al. 2021.

[0188] In some embodiments, provided herein are methods for determining or having determined a likelihood of response to a cancer treatment, wherein the treatment induces cytotoxicity through the RIPK3 pathway. For example, by increasing necroptosis, by derepression of TRIM28, by RIPK3 induction of cytokines and chemokines, etc (see, e.g. Zhou et al. (2024), Park et al. (2021), and Liu et al. (2021)).

[0189] In some embodiments, the RIPK3 pathway induces cytotoxicity of cancer cells by killing cancer cells via necroptosis; inducing T-cell-mediated cancer immune surveillance; secreting tumour-repressive cytokines; and / or restricting tumour development by inducing mitochondrial metabolism and ROS production. Necroptosis has been proposed as an alternative strategy for killing apoptosis-resistant cancer cells. In addition, due to the immunogenicity of necroptotic cells, induction of necroptosis in cancer cells has been used as a "vaccine'’ to trigger specific anti-cancer T cell responses. However, in some cancers, RIPK3 -signalling promotes cancer development and progression, and inhibition of RIPK3 signalling is a useful strategy to treat such cancers. Many RIPK3 inhibitors have been developed and are currently being used in clinical trials for autoimmune and inflammatory diseases, including cancers. Accordingly, in one embodiment, the cancer treatment involves overcoming apoptotic resistance in cancer cells by inducing necroptosis via the RIPK3 pathways. In another embodiment, the treatment involves inhibiting the RIPK3 pathway to induce cytotoxicity and cancer inhibition.

[0190] In some embodiments, subjects with samples that are RIPK3 positive are likely to respond to treatment. Conversely, subjects with samples that are not RIPK3 positive are not likely to respond to treatment.

[0191] 180539633.1Procaspase 8 effector pathway

[0192] In the extrinsic apoptotic pathway, the procaspase 8 is recruited by its death effector domains (DEDs) to the death-inducing signalling complex (DISC) to initiate extrinsic apoptosis execution. A membrane receptor complex is thereafter formed following the ligation of a member of the tumour necrosis factor receptor (TNFR) family. Procaspase 8 also inhibits necroptosis. Dysfunctional caspase 8 may contribute to the development of cancers.

[0193] In some embodiments, provided herein are methods for determining or having determined a likelihood of response to a cancer treatment, wherein the treatment induces cytotoxicity through the Procaspase 8 effector pathway. Killing of tumour cells by diverse cytotoxic approaches such as anticancer drugs, / -irradiation, suicide genes, or immunotherapy is mediated through induction of apoptosis in target cells. Activation of the cascade of proteolytic enzymes known as caspases is a critical component of the execution phase of cell death in most forms of apoptosis. Most signalling pathways activated by anticancer drugs ultimately result in activation of caspases. Many conventional cancer therapies induce apoptosis to remove the cancer cell by engaging caspases indirectly, though new er therapies aim to repress natural inhibitors of caspases. These include gene therapy and small molecule approaches. (For a recent review see, e.g. Boice and Bouchier-Hayes 2020.) Accordingly, in some embodiments, the cancer treatment is chemotherapy, anticancer drugs, / -irradiation, suicide genes, immunotherapy, gene therapy, and / or small molecules.

[0194] In some embodiments, subjects with samples that are procaspase 8 positive are likely to respond to treatment. Conversely, subjects with samples that are not procaspase 8 positive are not likely to respond to treatment.

[0195] Hedgehog signalling pathway

[0196] The Hedgehog (Hh) signalling pathway refers to a critical pathway involved in embryogenesis and the regulation of cell renewal and survival. It plays a significant role in regulating the cancer stem cell niche in several human cancers, including cancers with adnexal involvement and / or adnexal differentiation. The Hh signalling pathway is know n to contribute to cancer invasiveness and metastasis. Notch and Sonic Hh facilitate neovascularisation, angiogenesis, and epithelial-mesenchymal transition, and contribute to the maintenance of highly -metastatic tumour stem cells.

[0197] A dominant feature of all BCCs is the dysregulation of Hh signalling pathw ay. In the absence of ligand, Hh signalling is normally silenced by Patchedl (PTCHI), which inhibits Smoothened (SMO), an upstream activator of the signalling pathway. Upon

[0198] 180539633.1binding Hh ligands, PTCHI is inhibited, enabling SMO to transduce downstream signals via Glioma-Associated Oncogene Homolog 1 (GLI) transcription factors. In BCCs, this balance of PTCHI -mediated silencing, and SMO-mediated activation, is perturbed in ~80%-90% of tumours.

[0199] Despite the efficacy of Hh pathway inhibitors, they rarely eliminate all tumour cells and residual tumour cells can be detected in skin biopsies. Additionally, nearly all Gorlin BCCs will reappear at their original sites following discontinuation with current Hh inhibitors, but this may also reflect the germ-line mutations that predisposes to BCCs in patients with this syndrome.

[0200] Vismodegib binds to and inhibits SMO thereby preventing Hh signal transduction. In a clinical study testing vismodegib versus placebo in 41 BCNS patients, vismodegib administered orally at a dose of 150 mg daily showed a clinically significant reduction in new, surgically eligible BCCs and a reduction in existing BCC size. However, 54% of patients receiving vismodegib discontinued treatment due to unacceptable adverse events. An open-label, single arm study of vismodegib in 99 evaluable patients with advanced BCC including patients with Gorlin syndrome was conducted with patients dosed once daily with 150 mg. At this dose, 10 of 33 (30.3%) metastatic BCC patients showed an Objective Response Rate (ORR) with 27 of 63 (42.9%) locally advanced BCC patients exhibiting ORR. Objective Response (OR) was defined as a complete or partial response determined on two consecutive assessments separated by at least 4 weeks. At the 12-month update 33.3% metastatic BCC and 47.6% locally advanced BCC patient were responders.

[0201] There are two approved Hh pathway inhibitors available on the market in Australia. These are vismodegib (Erivedge®) and sonidegib (Odomzo®). Vismodegib is currently indicated for the treatment of adult patients with metastatic basal cell carcinoma, or with locally advanced BCC where surgery and / or radiation therapy are not appropriate (Erivedge® PI). The median duration of treatment with vismodegib is approximately 10 months. The most common adverse reactions (incidence of > 10%) are muscle spasms, alopecia, dysgeusia, weight loss, fatigue, nausea, diarrhoea, decreased appetite, constipation, arthralgias, vomiting, and ageusia. Many patients interrupt treatment due to AEs. Muscle spasms are typically the most frequently reported AE and occurs considerably early on in treatment. The median time to onset of muscle spasms is two months.

[0202] An additional rationale for treatment with vismodegib is that it is able to attenuate immune suppression in the tumour microenvironment, improve immunogenicity and

[0203] 180539633.1increase infiltration of T cells within 4 weeks of commencing treatment allowing for 4 weeks of treatment to be initially investigated in this study.

[0204] Hedgehog Pathway Inhibitors and ASN-002 both promote adaptive immune responses in BCC in a complementary manner. A major feature of IFNy therapy is tumour cell cycle arrest, apoptosis, anti-angiogenic effects and the induction of antitumour immunity. Hedgehog pathway inhibitors disrupt immune privilege within the tumour, thereby enabling an increased influx of cytotoxic T cells, and the release of intra-tumoural chemokines and cytokines. In addition, Suppressor of Cytokine Signalling 1. a negative regulator of interferon signalling which is induced after exposure of cells to Interferons (IFN), is blocked with the use of Hh pathway inhibitors.

[0205] The use of Hh pathway inhibitors with ASN-002 is expected to be synergistic and allow for lower doses of ASN-002 to be used with equivalent or superior efficacy. By reducing the dose of ASN-002, up to 6 BCC tumours per treatment cycle may be treated. In addition, reducing the duration of vismodegib treatment to 4 weeks (versus 7 to 8 months) improves the safety and tolerability and would assist w ith treatment compliance.

[0206] The combination use of Hh pathway inhibitors plus ASN-002 is an efficacious alternative option to surgical excision, with an acceptable safety’ profile, for patients with multiple BCCs and could be used to concurrently treat multiple tumours during multiple clinical visits during the course of a year.

[0207] In some embodiments, the treatment comprises administering a therapeutically effective amount of a hedgehog signalling pathway inhibitor.

[0208] In some embodiments the at least one agent that inhibits the Hedgehog (Hh) signalling pathway inhibits the agent that inhibits the Hh signalling pathway inhibits a target selected from among SMO, PTCHI, GLI, SHHat, tGLIl, and SHH. In some embodiments the Hh signalling pathway target is SMO. In other embodiments the Hh signalling pathway target is SHH.

[0209] In some embodiments the agent that inhibits the Hh signalling pathway is a small molecule inhibitor. In some embodiments the small molecule inhibitor is selected from the group consisting of: Vismodegib, Sonidegib, Saridegib, IPI 926, LEQ-506, Taladegib, Itraconazole, Glasdegib, Jervine, CUR61414, BMS-833923, TAK-441, MRT-92, GDC -0449, HH-13, GANT61, and HH-20. In some preferred embodiments the small molecule inhibitor is Vismodegib. In some embodiments the small molecule inhibitor of the Hh signalling pathway is administered at a dose of about 150 mg to about 500 mg per day. In some embodiments the small molecule inhibitor of the Hh signalling pathway is administered at a dose of about 150 mg / day.

[0210] 180539633.1In other embodiments the agent that inhibits the Hh signalling pathway comprises a polypeptide, a polynucleotide, or a peptide.

[0211] Tn some embodiments the agent that inhibits the Hh signalling pathway comprises an antibody or an antigen-binding portion thereof that binds specifically to an Hh signalling pathway target. In some embodiments the agent comprises an antigen-binding portion against SHH, SMO, PTCHI, GLI, SHHat, or tGLIl. In some embodiments the agent comprises an antibody or an antigen-binding portion thereof against SHH. In some embodiments the antibody or antigen-binding portion thereof against SHH is an antibody or antigen-binding portion thereof selected from the group consisting of 5E1, MEDI-5304, 1C11-2G4, antigen-binding portions thereof, and antibodies or antigen-binding portions thereof that compete with any one of 5E1, MEDI-5304. or 1C11-2G4 for binding to SHH. In other embodiments the agent comprises an antibody or an antigenbinding portion thereof against PTCHI. In some embodiments the antibody against PTCHI is the a-PTCHl antibody.

[0212] In some embodiments the agent that inhibits the Hh signalling pathway is a polynucleotide. In some embodiments the polynucleotide encodes a polypeptide that inhibits the Hh signalling pathway. In some embodiments, the polynucleotide is or encodes a miRNA, an siRNA, an RNAi, or a CRISPR gRNA, an antisense RNA, or an antisense oligonucleotide targeted against a Hh signalling pathway target.

[0213] Recombinant virus

[0214] In some embodiments, the treatment comprises administering a therapeutically effective of a recombinant virus comprising an expression cassette encoding one or more biotherapeutic agents. Recombinant viruses are those that are generated to incorporate modification(s) introduced into a specific gene / locus (or multiple loci) of the viral genome. Recombinant viruses can be used to genetically modify cells, triggering the expression of tumour-specific antigens. These viruses can also be used to directly infect antigen-presenting cells to boost anti-cancer immune responses.

[0215] Suitable types of DNA viruses include adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), retrovirus, and lentivirus. Methods for design, production, and use of such types of recombinant DNA viruses are established in the art, as exemplified in Fukazawa et al. (2010) and in “Gene Therapy Protocols” for adenovirus; “Adeno- Associated Virus: Methods and Protocols” for AAV; Cody et al. (2013) and “Herpes Simplex Virus: Methods and Protocols” for HSV; “Gene Therapy Protocols Vol. 1 : Production and In Vivo Applications of Gene Transfer Vectors” and Amer et al. (2014) for retrovirus; and Merten et al. (2016) and Emeagi et al. (2013) for

[0216] 180539633.1lentivirus. In some preferred embodiments, the recombinant virus to be used in the treatment method is an adenovirus. In some preferred embodiments the recombinant adenovirus is ASN-002, a replication-deficient type 5 adenovirus for expression of interferon gamma (also known as Tgl042 and SP-002) (Urosevic, 2007; Liu et al., 2004; Dummer et al., 2004 and 2010; Accart et al., 2013; Khammari et al., 2015; Dreno et al., 2014; Hillman et al., 15 2004).

[0217] In some embodiments the biotherapeutic agent to be expressed is selected from the group consisting of: cytokines, chemokines, chemokine antagonists, chemokine receptor antagonists, costimulatory molecules, and antibodies. In some embodiments the biotherapeutic agent to be expressed is the cytokine interferon gamma.

[0218] In one example, interferon- / (IFN-y) plays a key role in activation of cellular immunity and subsequently, stimulation of antitumor immune-response. Based on its cytostatic, pro-apoptotic and antiproliferative functions, IFN-y is considered potentially useful for adjuvant immunotherapy for different types of cancer. Moreover, IFN-y may inhibit angiogenesis in tumour tissue, induce regulatory T-cell apoptosis, and / or stimulate the activity’ of Ml proinflammatory macrophages to overcome tumour progression.

[0219] Accordingly, in some embodiments, treatment comprises a recombinant virus for expression of IFN-y in a subject. In some embodiments, the recombinant virus is a recombinant adeno-associated virus (AAV). In some embodiments, the recombinant DNA virus comprises an expression cassette encoding IFN-y. In some embodiments, an expression cassette encoding IFN-y is operable for expression in mammalian cancer cells. In some embodiments, where the biotherapeutic agent to be expressed is interferon gamma, and the recombinant virus is the recombinant adenovirus ASN-002 (SP-002)-see, e.g., Clinical Trial NCT 02550678. ASN-002, also known as Gusacitinib (CAS No.: 1425381-60-7), is an orally active dual SYK / JAK kinase inhibitor. Gusacitinib rapidly and significantly suppresses key inflammatory pathways implicated in atopic dermatitis pathogenesis. Gusacitinib can be used in the research of chronic hand eczema and cancers such as basal cell carcinoma. Clinical trials include a Phase Ila study to assess the efficacy and safety of ASN-002 alone and in combination with chemotherapy in adult participants with low-risk basal cell carcinomas (trial registration number ACTRN12619001298101).

[0220] In some embodiments, the drug is ANS-002 in combination with a hedgehog pathway inhibitor for example, vismodegib. See, e g. research study 2021.058.

[0221] In some embodiments the recombinant virus is a recombinant DNA virus. In some embodiments the recombinant DNA virus is an adenovirus, an adeno-associated

[0222] 180539633.1virus (AAV), a herpes simplex virus (HSV), or a lentivirus. In some preferred embodiments the recombinant DNA virus is an adenovirus. In some preferred embodiments the recombinant adenovirus is for expression of interferon gamma. In some preferred embodiments the adenovirus for expression of interferon gamma is ASN-002 / SP-002.

[0223] Checkpoint inhibitors

[0224] Immune checkpoints are a normal part of the immune system and prevent an immune response from being so strong that it destroys healthy cells in the body. Immune checkpoints engage when proteins on the surface of T cells recognise and bind to partner proteins on other cells, so called immune checkpoint proteins. When the checkpoint and partner proteins bind together, they send an “off” signal to the T cells. When the partner protein is a cancer or tumour protein rather than a ‘self protein, immune checkpoints can prevent the immune system from destroying the cancer. Accordingly, immune checkpoint inhibitors are a class of drug which prevent the protective effect afforded by immune checkpoints on cancer cells, commonly by blocking the binding of checkpoint proteins with tumour partner proteins, allowing the T cells to kill cancer cells. For example, use of monoclonal antibodies that target binding interactions that are important to checkpoints of immune activation.

[0225] Checkpoint inhibitor proteins commonly targeted by drugs include CTLA-4 (cytotoxic T lymphocyte associated protein 4), PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death ligand 1). CTLA-4 and PD-1 are found on T cells. PD-L1 are on cancer cells.

[0226] Immune checkpoint inhibitors are currently approved to treat a variety of cancer types, including: skin cancers, including BCC and melanoma; breast cancer; bladder cancer; cervical cancer; colon cancer; head and neck cancer; Hodgkin lymphoma; liver cancer; lung cancer; renal cell cancer (a type of kidney cancer); stomach cancer; rectal cancer; and any solid tumour that is not able to repair errors in its DNA that occur when the DNA is copied.

[0227] In some embodiments, the treatment method includes immunotherapy against a checkpoint protein, for example, administration of an immune checkpoint inhibitor. Examples of suitable immune checkpoint inhibitors to be administered include, but are not limited to an antibody against Programmed Death (PD-1), an antibody against PD-Ligand 1 (PDL-1), an antibody against Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4), an antibody against Adenosine A2A receptor (A2AR), an antibody against B and T Lymphocyte Attenuator (BTLA), an antibody against Cytotoxic T-Lymphocyte-

[0228] 180539633.1Associated protein 4 (CTLA-4), an antibody against indoleamine 2,3-dioxygenase (IDO), an antibody against Lymphocyte Activation Gene-3 (LAG3), and an antibody against T-cell immunoglobulin domain, Mucin domain 3 (TIM-3), an antibody against killer-cell immunoglobulin-like receptor (KIR), an antibody against CD94, an antibody against Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), antibodies against any of Leukocyte immunoglobulin-like receptor subfamily A (LILRA) members 1-6 (LILRA 1-LILRA6), antibodies against any of Leukocyte immunoglobulin-like receptor subfamily B (LILRB) members 1-5, or any combination thereof.

[0229] In some embodiments, the checkpoint inhibitor is selected from the group consisting of an inhibitor of PD-1, CTLA-4, and PD-L1. For example, the checkpoint inhibitor is an antibody. For example, the checkpoint inhibitor is an anti-PD-1 antibody. For example, the checkpoint inhibitor is Pembrolizumab, Nivolumab, and / or Cemiplimab. For example, the checkpoint inhibitor is an anti-CTLA-4 antibody. For example, the checkpoint inhibitor is Ipiliumab. For example, the checkpoint inhibitor is an anti-PD-Ll antibody. For example, the checkpoint inhibitor is Atezolizumab, Avelumab, and / or Durvalumab. For example, the checkpoint inhibitor targets CTLA-4 protein. For example, the checkpoint inhibitor targets PD-1 protein. For example, the checkpoint inhibitor targets PD-L1 protein. For example, the checkpoint inhibitor targets CD80. For example, the checkpoint inhibitor targets CD86. For example, the checkpoint inhibitor targets GTPase KRas. For example, the checkpoint inhibitor targets cytochrome P450 family protein. For example, the checkpoint inhibitor targets ABCB1 protein. For example, the checkpoint inhibitor targets lymphocyte activation gene 3 protein. For example, the checkpoint inhibitor targets 5'-nucloetidase protein. For example, the checkpoint inhibitor is Quemliclustat. Relatimab, Adagrasib, Sotorasib, Belatacept, Abatacept. Tremelimumab, Anti-OX40 antibody BMS 986178, CA-170, AUNP-12, Cosibelimab, Envafolimab, Durvalumab, Avelumab, Atezolizamab, MEDI0680, AMP-224, Retifanlimab, Dostarlimab, Toripalimab, Tislelizumab, Sintilimab, Camrelizumab, Spartalizumab, Cemiplimab, Prmbrolizumab, Nivolumab, and / or Ipilimumab.

[0230] Treatment with ASN-002

[0231] Intra-tumoural injections of interferons have been shown to be an effective treatment for non-melanoma skin cancers, with remission rates of 70-100% in BCC patients. Recombinant adenovirus vectors (rAd) have been previously utilised to deliver therapeutic or prophylactic agents, including interferons. In particular, the rAd ASN-002 (an adenoviral vector encoding interferon y [IFN y]) has previously been shown to be well-tolerated and have clinical activity, inducing objective tumour responses after intra-

[0232] 180539633.1tumoural injection in patients with BCC, cutaneous T-cell lymphoma and primary cutaneous B-cell lymphoma. ASN-002 is replication deficient and although it infects cells, it is not able to replicate in the tumour or in normal human cells. Infected cells, however, are able to transcribe and translate the IFN y DNA, leading to a sustained, effective local concentration of IFNy in the tumour microenvironment but avoids serum levels of IFNy that may lead to unacceptable systemic toxicity.

[0233] A Phase l / 2a study of ASN-002 (ASN-002-001) in adult patients with low-risk nodular BCC (nBCC) was conducted in Australia. Fifteen eligible participants were accrued to this trial in three dose escalation cohorts. Each participant received one injection of intra-tumoural ASN-002 per week (Weeks 1, 2, and 3) into the target tumour that was then excised at week 16-17. The initial cohort (n=3) received 5 x 1010.

[0234] ASN-002 viral particles (vp) / inj ection, the second cohort (n=6) received 1.5 x 1011 vp / injection, and the third cohort (n=6 eligible participants) received 3.0 x 1011 vp / inj ection.

[0235] Safety and BCC response data were encouraging. Toxicity was largely limited to Grade 1-2 local skin responses (erythema, induration, ulceration and pain) at the ASN-002 injection site in all participants and Grade 1 flu-like symptoms (headache, malaise, and fever) in 7 of 12 participants in the higher two dose cohorts and Grade 2 flu-like symptoms in 2 of 6 eligible participants in the highest dose cohort. Flu-like symptoms persisted for only 1-2 days after injection, with severity and duration of symptoms decreased after the second and third injections of ASN-002 compared to the initial injection. Importantly, maximum tolerated dose was not achieved at 3.0 x 1011vp / injection and no dose limiting toxi cities observed.

[0236] Histological clearance (pathological) complete response (CR) was observed in 1 of 3 participants in the low dose cohort (33%), 5 of 6 participants in the intermediate cohort, and 5 of 6 participants in the high dose cohort, i.e. a histological CR was seen in 10 / 12 participants (83%) treated at the higher two doses. In addition, abscopal regression was observed in the majority of non-injected tumours. The abscopal responses are thought to be mediated by the induction of a systemic cytotoxic T cell response that is elicited during intra-tumoural ASN-002 treatment (see ASN-002, Investigator's Brochure, Edition 5.0, Dated 6Apr2020. 2020, Ascend Bipharmaceuticals Ltd).

[0237] Formulation and use of Therapeutic Agents

[0238] Provided herein is a use of a therapeutic agent in the manufacture of a medicament for treating cancer in a subject determined to have a likelihood of a response to the

[0239] 180539633.1therapeutic agent using a method described herein. For example, the cancer is basal cell carcinoma.

[0240] Also provided herein is a therapeutic agent for use in treatment of cancer in a subject determined to have a likelihood of a response to the therapeutic agent using a method described herein. For example, the cancer is basal cell carcinoma.

[0241] Any of the therapeutic agents described herein can be formulated either alone or in combined pharmaceutical compositions as described herein for administration to a subject via any conventional means including, but not limited to, intralesional, parenteral (for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or intrapleural), oral, or transdermal administration routes.

[0242] Therapeutic agents can be formulated into any suitable dosage form, including but not limited to, injectable formulations, aqueous oral dispersions, liquids, mists, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a patient to be treated, solid oral dosage forms, controlled release formulations, lyophilised formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

[0243] In some embodiments, the dose comprises 5 x 10(11) virus particles (vp), 1.5 x 10(1 1) vp, 3.0 x 10(11) vp, 2.25 x 10(11) vp, 3 x 10(9) vp, or 3 x 10(10) vp. In some embodiments, the dose is administered by injection. In some embodiments, the dose is given weekly for three weeks. For example, the dose is 5 x 10(11) virus particles (vp), weekly injection for 3 weeks. For example, the dose is 1.5 x 10(11) vp) weekly injection for 3 weeks. For example, the dose is 3.0 x 10(11) vp weekly injections for 3 weeks. For example, the dose is 2.25 x 10(11) vp weekly injections for 3 weeks. In some embodiments, the dose comprises 5 x 10(11) virus particles (vp) & 5-FU (5mg). In some examples, ASN-002 is administered together with 5-FU at 1 mg, 2.5 mg, 5 mg, 10 mg or 25 mg.

[0244] In some embodiments, the dose comprises 10, 20, 30, 40, 50, or 75 mg twice daily. In some embodiments, the dose comprises 80 or 120 mg daily. For example, the dose comprises ASN-002.

[0245] In some embodiments, treatment is given weekly for 3 to 10 weeks. For example, weekly for 3 weeks. For example, weekly for four weeks. For example, weekly for five weeks. For example, weekly for six weeks. For example, weekly for seven weeks. For example, weekly for eight weeks. For example, weekly for nine weeks. For example, weekly for ten weeks.

[0246] 180539633.1In some embodiment, the volume of the dose is dependent on the size of the tumour. For example, a tumour of less than 6 mm, will receive a volume of 0.5 mL. For example, a tumour of 6-10 mm will receive a volume of 1 mL. For example, a tumour of 11-20 mm will receive a volume of 1.5 mL.

[0247] In some embodiments, does are split evenly between two or more administrations. In some embodiments, the doses are uneven.

[0248] Rationale for ASN-002 dose selection: Previously, the use of ASN-002 as a monotherapy at 5xl010vp / injection resulted in a histological clearance rate of 33% and was well tolerated. It was also demonstrated that 3xl0nvp / injection can be administered without any serious adverse events (SAE). Hence, at the low dose, up to 6 tumours may be injected without any SAEs anticipated. The use of Hh pathway inhibitors generally result in the onset of adverse events (AE) after 6 weeks of continued therapy while the immune-potentiating effects can be observed after 4 weeks of treatment, hence the use of ASN-002 will be initially evaluated with the use of Hh for a 4-week Hh treatment period.

[0249] Previously, the use of ASN-002 as a monotherapy at 1.5xl0nvp / injection resulted in an 83% histological clearance rate, as well as demonstrating abscopal regression in over 70% of non-target BCCs. These responses included both complete and partial responses. The use of the combination modality with their complementary and synergistic mechanisms of action will also be evaluated for the prospect of further improved responses in non-target tumours. It is anticipated that the synergistic combination effect of ASN-002 and vismodegib may allow for a dose reduction of ASN-002 and earlier discontinuation of vismodegib therapy.

[0250] ASN-002 (formerly TG-1042, aka AD-IFNy has been the subject of several trials, including as described in Dummer et al. (2010), TGN-002.06, and ClinicalTrials.gov Identifier: NCT02550678.

[0251] Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with one or more of the therapeutic agents described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatine, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents may be added, such as the cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0252] 180539633.1Pharmaceutical solid dosage forms can include, in addition to the therapeutic agents, one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavouring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, antifoaming agent, antioxidant, preservative, or one or more combination thereof.

[0253] Suitable carriers for use in the solid dosage forms described herein include, but are not limited to, acacia, gelatine, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol and the like.

[0254] Suitable fdling agents for use in the solid dosage forms described herein include, but are not limited to, lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulphate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinised starch, hydroxypropylmethycellulose (HPMC), hydroxypropylmethycellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0255] In order to release the therapeutic agents from a solid dosage form matnx as efficiently as possible, disintegrants are often used in the formulation, especially when the dosage forms are compressed with binder. Disintegrants help rupturing the dosage form matrix by swelling or capillary action when moisture is absorbed into the dosage form. Suitable disintegrants for use in the solid dosage forms described herein include, but are not limited to, natural starch such as com starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as a wood product, methylciystalline cellulose, for example,, Avicel® PH101, Avicel®PH102, Avicel®PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a crosslinked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum® EIV (magnesium aluminium silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch

[0256] 180539633.1glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulphate, sodium lauryl sulphate in combination starch, and the like.

[0257] Binders impart cohesiveness to solid oral dosage form formulations: for powder filled capsule formulation, they aid in plug formation that can be filled into soft or hard shell capsules and for tablet formulation, they ensure the tablet remaining intact after compression and help assure blend uniformity prior to a compression or fill step. Materials suitable for use as binders in the solid dosage forms described herein include, but are not limited to, carboxymethylcellulose, methylcellulose (for example,, Methocel®), hydroxypropylmethylcellulose (for example, Hypromellose USP Pharmacoat-603, hydroxypropylmethylcellulose acetate stearate (Aqoate HS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (for example,, Klucel®), ethylcellulose (for example,, Ethocel®), and microcrystalline cellulose (for example,, Avicel®), microcrystalline dextrose, amylose, magnesium aluminium silicate, polysaccharide acids, bentonites, gelatine, polyvinylpyrrolidone / vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, a sugar, such as sucrose (for example, Dipac®), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (for example, Xylitab®), lactose, a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, starch, polyvinylpyrrolidone (for example,, Povidone® CL, Kollidon® CL, Polyplasdone®XL-10, and Povidone®K-12), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodium alginate, and the like.

[0258] In general, binder levels of 20-70% are used in powder-fdled gelatine capsule formulations. Binder usage level in tablet formulations varies whether direct compression, wet granulation, roller compaction, or usage of other excipients such as fdlers which itself can act as moderate binder. Formulators skilled in art can determine the binder level for the formulations, but binder usage level of up to 70% in tablet formulations is common.

[0259] Suitable lubricants or glidants for use in the solid dosage forms described herein include, but are not limited to, stearic acid, calcium hydroxide, talc, com starch, sodium steary 1 fumarate, alkali-metal and alkaline earth metal salts, such as aluminium, calcium, magnesium, zinc, stearic acid, sodium stearates, magnesium stearate, zinc stearate, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol or a methoxypolyethylene glycol such as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glyceryl behenate,

[0260] 180539633.1glyceryl palmitostearate, glyceryl benzoate, magnesium or sodium lauryl sulphate, and the like.

[0261] Suitable diluents for use in the solid dosage forms described herein include, but are not limited to, sugars (including lactose, sucrose, and dextrose), polysaccharides (including dextrates and maltodextrin), polyols (including mannitol, xylitol, and sorbitol), cyclodextrins and the like.

[0262] Suitable wetting agents for use in the solid dosage forms described herein include, for example, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, quaternary ammonium compounds (for example,, Polyquat 10®), sodium oleate, sodium lauryl sulphate, magnesium stearate, sodium docusate, triacetin, vitamin E TPGS and the like.

[0263] Suitable surfactants for use in the solid dosage forms described herein include, for example, sodium lauryl sulphate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glycery l monostearate, copolymers of ethylene oxide and propylene oxide, for example,, Pluronic® (BASF), and the like.

[0264] Suitable suspending agents for use in the solid dosage forms described here include, but are not limited to, polyvinylpyrrolidone, for example,, polyvinylpyrrolidone KI 2, polyvinylpyrrolidone KI 7, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyethylene glycol, for example,, the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, vinyl pyrrolidone / vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose. polysorbate-80. hydroxyethylcellulose, sodium alginate, gums, such as, for example,, gum tragacanth and gum acacia, guar gum, xanthans. including xanthan gum, sugars, cellulosics. such as, for example,, sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxy ethylcellulose, polysorbate-80, sodium alginate, poly ethoxylated sorbitan monolaurate, poly ethoxylated sorbitan monolaurate, povidone and the like.

[0265] It should be appreciated that there is considerable overlap between additives used in the solid dosage forms described herein. Thus, the above-listed additives should be taken as merely exemplary, and not limiting, of the ty pes of additives that can be included in solid dosage forms described herein. The amounts of such additives can be readily determined by one skilled in the art, according to the particular properties desired.

[0266] 180539633.1Liquid formulation dosage forms for oral administration can be aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.

[0267] The aqueous suspensions and dispersions described herein can remain in a homogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005 edition, chapter 905), for at least four hours. The homogeneity should be determined by a sampling method consistent with regard to determining homogeneity of the entire composition. In one embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 1 minute. In another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 30 seconds. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion.

[0268] In addition to the additives listed above, the liquid formulations can also include inert diluents commonly used in the art, such as water or other solvents, solubilising agents, and emulsifiers. Exemplary emulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodium doccusate, cholesterol, cholesterol esters, taurocholic acid, phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, com germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, or mixtures of these substances, and the like.

[0269] Injectable formulations

[0270] Formulations suitable for intramuscular, subcutaneous, or intravenous injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection may also contain additives such as preserving, wetting, emulsifying, and dispensing agents.

[0271] 180539633.1Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminium monostearate and gelatine.

[0272] For intravenous injections, therapeutic agents described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, appropriate formulations may include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are generally known in the art.

[0273] Parenteral injections may involve bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the therapeutic agents in water-soluble form. Additionally, suspensions of the therapeutic agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity’ of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilisers or agents which increase the solubility’ of a therapeutic agent to allow for the preparation of highly concentrated solutions. Alternatively, the therapeutic agent may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. The therapeutic agents described herein may be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more therapeutic agents. The unit dosage may be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multiple-dose

[0274] 180539633.1reclosable containers can be used, in which case it is typical to include a preservative in the composition. By way of example only, formulations for parenteral injection may be presented in unit dosage form, which include, but are not limited to ampoules, or in multidose containers, with an added preservative.

[0275] EXAMPLES

[0276] Example 1. Clinical pathology results

[0277] Materials and Methods

[0278] Sectioning of FFPE blocks and mounting on slides

[0279] 1. Trim off any excess paraffin if necessary. Block face should be optimally square or rectangle.

[0280] 2. Use a pencil and label the slides with block number and slide number.

[0281] 3. Place the block on the locked microtome chuck such that the wax block faces the blade and is aligned in a vertical plane.

[0282] 4. Trim off excess paraffin to expose the entire cutting surface of the tissue.

[0283] 5. Chill the block face down on cold plate using a little amount of distilled water in a clean dish.

[0284] 6. Put the block back on the chuck, carefully advance the block with the hand wheel and cut sections at thickness of 3-4um or as required. The knife angle will be 10 degrees.

[0285] 7. Pick the ribbon of sections with a curved forceps and transfer to a water bath with distilled water at 40 to 450C.

[0286] 8. Pick up the expanded section on a clean, labelled slide.

[0287] 9. Air dry the slides overnight at room temperature.

[0288] H&E staining

[0289] The slides with mounted sections are loaded into an auto stainer and the following programmed sequence is used.

[0290] 1. Xylene 3min

[0291] 2. Xylene 3min

[0292] 3. Xylene 3min

[0293] 4. Ethanol Imin

[0294] 5. Ethanol Imin

[0295] 6. Ethanol Imin

[0296] 7. 70% Ethanol 30secs

[0297] 8. Water Imin

[0298] 9. Haematoxylin 4min (accurate step)

[0299] 180539633.110. Water Imin

[0300] 11. Water Imin

[0301] 12. Scott’s tap water 45sec

[0302] 13. Water 2min

[0303] 14. Eosin 4min (accurate step)

[0304] 15. Water 15sec

[0305] 16. Ethanol 45 sec

[0306] 17. Ethanol 45 sec

[0307] 18. Ethanol 45 sec

[0308] 19. Ethanol Imin

[0309] 20. xylene 2min

[0310] 21. Xylene 3 min

[0311] 22. Xylene 3min

[0312] 23. Exit.

[0313] Clinical trial protocol

[0314] Study drug(s): ASN-002; Vismodegib.

[0315] This study aims to test the dose of l.OxlO11VP / injection in combination with standard vismodegib therapy for 4 weeks. Following recruitment and pending safety review of the data from Arms 1 and 2 (Table 2), additional treatment Arms may be tested.

[0316] The primary objectives are to:

[0317] 1) Evaluate the safety and tolerability of intra-tumoural ASN-002 when administered in combination with oral vismodegib or as a monotherapy in patients with BCCs;

[0318] 2) Evaluate the efficacy’ of intra-tumoural ASN-002 in target tumours when administered in combination with oral vismodegib or as a monotherapy in patients with BCCs.

[0319] The secondary objective is to:

[0320] 1) Evaluate the efficacy of intra-tumoural ASN-002 in non-target tumours when administered in combination with oral vismodegib or as a monotherapy in patients with BCCs.

[0321] The exploratory objective is to:

[0322] 1) Evaluate immunological biomarkers during the course of treatment.

[0323] 180539633.1Methodology

[0324] This study will evaluate ASN-002 (in the dose range 0.5 to 1.5xl0nvp / rnL) with the Hh inhibitor vismodegib (Erivedge®). The study will initially evaluate two Arms receiving 1.0 x 1011vp / injection, and following a safety review, may implement further arms in an adaptive study design.

[0325] Following screening and baseline biopsies for target and non-target tumours, eligible subjects will be enrolled in the study.

[0326] Cycle 1: Treatment with vismodegib (daily dose of 150 mg) for 4 weeks and ASN-002 for 3 weeks (i.e., three ASN-002 injections in total):

[0327] • Day 1 to Day 14 - vismodegib alone

[0328] • Day 15 - vismodegib and ASN-002

[0329] • Day 16 to Day 21 - vismodegib alone

[0330] • Day 22 - vismodegib and ASN-002

[0331] • Day 23 to Day 28 - vismodegib alone

[0332] • Day 29 - ASN-002 alone

[0333] Tumour evaluation will be performed at Week 17, following which, the investigator will initiate Cycle 2 if residual tumour remains (definite or probable).

[0334] Cycle 2: Treatment with vismodegib (daily dose of 150 mg) for 4 weeks, and one further injection with ASN-002:

[0335] • Day 1 to Day 7 - vismodegib alone

[0336] • Day 8 - vismodegib and ASN-002

[0337] • Day 9 to Day 28 - vismodegib alone

[0338] Surgical excision for all patients will occur at Week 25 or Week 33 at the investigators' discretion, and dependent on when patient completed study treatment (1 or 2 treatment cycles). Up to 10 BCCs to be excised including 3 target tumours. Excisions can be conducted over 2 visits as per Investigator’s discretion.

[0339] Table 2; Arm allocation and dosing schedule

[0340]

[0341] 180539633.1

[0342]

[0343] 1 Where a second cycle of treatment is indicated, only one injection of ASN-002 will be given.

[0344] 2 Vismodegib dosing will be for a minimum of 4 weeks with a subsequent 4 weeks at Investigators discretion following the week 17 visit.

[0345] 3 Patients with 2 (confirmed eligible) BCCs may be considered for entry to this treatment arm on a case-by-case basis, following consultation between the investigator, medical monitor and sponsor.

[0346] ARM 3 to ARM 8 - adaptive design arms. At the discretion of the Safety7Review Committee, alternative doses of ASN-002 may be explored in the adaptive design arms (ASN-002 in the dose range 0.5xl0nvp or I.5xl0nvp) in combination with 150 mg vismodegib administered daily for 4 weeks. Vismodegib or ASN-002 may be evaluated as monotherapies to provide control groups to allow comparison of treatment Arms. Initially, 18 subjects with 2 target tumours will be randomized 1:1:1 to the following treatment groups: Arm 8A: 1.5 xlOl 1 vp ASN-002 / target tumour (qWk x 3) x 3 Cycles; Arm 8B: 0 xlOl 1 vp ASN-002 / target tumour (Placebo) (qWk x 3) x 1 Cycle; Arm 8C: 1.5 xlOl 1 vp ASN-002 / target tumour (qWk x 3) x 1 Cycles.

[0347] Clinical pathology results from the trial are summarised in Tables 3 and 4 below. Acrosyringia can be considered as a subset of eccrine / apocrine carcinomas. Ductal carcinoma is a subtype of eccrine / apocrine cancers. Skin adnexal tumours / sweat gland tumours range from small duct lumens within individual tumour cells to large dilated cystic space that can comprise the entire tumour volume.

[0348] Table 3: Summary of clinical data

[0349]

[0350] 180539633.1

[0351]

[0352] 180539633.1

[0353]

[0354] Table 4: Clinical pathology results

[0355] &

[0356] &

[0357]

[0358] 180539633.1

[0359]

[0360] &

[0361] &

[0362]

[0363] 180539633.1

[0364]

[0365] &

[0366] &

[0367] &

[0368] &

[0369] &

[0370]

[0371] 180539633.1

[0372]

[0373]

[0374] 180539633.1

[0375]

[0376]

[0377] 180539633.1Example 2. Determination of a likelihood of response to cancer treatment and patient stratification

[0378] In order to determine whether there was a correlation between patients who responded well to cancer treatment and patients who did not, patient data from Table 4 was categorised and stratified to identify common factors of reliable and robust predictive value.

[0379] Histological assessment on samples stained with haematoxylin and eosin showed that patients with adnexal differentiation and / or adnexal involvement had a decrease likelihood of response to cancer treatment compared to patient whose samples did not display adnexal differentiation and / or adnexal involvement.

[0380] A biomarker panel was used to determine the cell cycle status of tumour cells, specifically the presence or absence of mitosis and / or cell proliferation. The results showed that where mitotic activity and / or cell proliferation is detected, patients had an increased likelihood of responding to cancer treatment compared to those whose samples did not demonstrate evidence of mitotic activity and / or cell proliferation.

[0381] Accordingly, histological assessment, mitotic activity and / or cell proliferation status, or a combination of histological assessment and mitotic activity and / or cell proliferation status reliably determines a likelihood of response to a cancer treatment. This enables, for example, identification of patients for whom treatment will more likely be of benefit, and conversely, patients who require additional management for their cancer.

[0382] The identified features were then used to stratify patients in a clinical trial based on how likely they are to have a positive response to a cancer treatment, and / or how likely they are to have residual disease once the treatment has been completed. The stratification identifies the highest rate of efficacy for each of the subjects treated. This enables, for example, selection of a clinical trial cohort of patients with similar prospects of response to treatment, removing another variable from the clinical trial setting to achieve more accurate and reliable clinical trial data.

[0383] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0384] 180539633.1All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0385] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0386] 180539633.1REFERENCES

[0387] Alsaad et al., (2007) J Clin Pathol. 60:129-44.

[0388] Amer et al. (2014) Molecular and Cellular Therapies 2:27.

[0389] Boice and Bouchier-Hayes (2020) BBA Mol. Cell Res. 1867:118688.

[0390] Cody et al. (2013) Journal of Genetic Syndromes & Gene Therapy 4: 126.

[0391] Dummer et al. (2010) Mol Ther, 2010. 18:1244-7.

[0392] Duronio and Xiong (2013) Cold Spring Harb Perspect Biol 5:a008904.

[0393] Emeagi et al. (2013) Current Molecular Medicine 13:602-625.

[0394] Givens et al. (2009) Am Fam Physician. 80:815-820.

[0395] Idriss et al. (2024) Cancer Lett. 589:216821.

[0396] Kazakov et al (2013) Cutaneous Adnexal Tumors, Wolters Kluwer Health.

[0397] Liu et al (2021) Cell Mol Life Sci 78:7199-7217.

[0398] Lopez-Knowles et al (2010) Cancer Epidemiol Biomarkers Prev 19: 301-309.

[0399] Merten et al. (2016) Molecular Therapy - Methods & Clinical Development 3:16017. Park et al. (2021) Cell Mol Life Sci. 78:7199-7217.

[0400] Shapiro et al (2013) Ann Surg 257: 548-554.

[0401] Wheeler et al. (2023) Am Fam Physician. 108:580-587.

[0402] Zhou et al. (2024) Cell Death Discov. 10:200.

[0403] 180539633.1

Claims

CLAIMS1. A method for determining or having determined a likelihood of response to a treatment of basal cell carcinoma in a subject, the method comprising detecting the presence or absence of adnexal differentiation and / or adnexal involvement in a skin lesion sample from the subject, and providing a result indicating an increased likelihood of response to the treatment if no adnexal differentiation and / or adnexal involvement is detected, or providing a result indicating a decreased likelihood of response to the basal cell carcinoma treatment if adnexal differentiation and / or adnexal involvement is detected.

2. The method of claim 1, wherein the skin lesion has been determined to be, or likely to be, basal cell carcinoma.

3. The method of claim 1 or claim 2, further comprising selecting the subject for a clinical trial for treatment of basal cell carcinoma if and only if no adnexal differentiation and / or adnexal involvement is detected, or excluding the subject from the clinical trial if and only if adnexal differentiation and / or adnexal involvement is detected.

4. The method of claim 1 or claim 2, further comprising selecting the subject for the treatment if and only if no adnexal differentiation and / or adnexal involvement is detected, or excluding the subject from the treatment if and only if adnexal differentiation and / or adnexal involvement is detected.

5. The method according to any one of claims 1 to 4, wherein detecting the presence or absence of adnexal differentiation and / or adnexal involvement comprises staining the sample.

6. The method of claim 5, wherein the stain is haematoxylin and eosin.

7. The method according to any one of claims 1 to 4, wherein detecting the presence or absence of adnexal differentiation and / or adnexal involvement comprising determining a level of uncomplexed P-catenin in the sample, wherein adnexal differentiation is associated with a higher level of uncomplexed P-catenin.180539633.

18. A method for determining or having determined a likelihood of response to a treatment of a cancer in a subject, the method comprising detecting the presence or absence of mitotic activity and / or cell proliferation in a sample from the subject, and providing a result indicating an increased likelihood of response to the treatment if mitotic activity and / or cell proliferation is detected, or providing a result indicating a decreased likelihood of response to the cancer treatment if mitotic activity and / or cell proliferation is not detected, wherein the treatment induces cytotoxicity through RIPK3 or procaspase 8 effector pathways.

9. The method of claim 8, wherein the sample is a skin lesion sample and the cancer is a cutaneous cancer.

10. The method of claim 9, wherein the skin lesion has been determined to be, or likely determined to be, a cutaneous cancer.

11. The method of claim 9 or claim 10, wherein the cutaneous cancer is basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, sebaceous carcinoma, primary epithelial cancers, colorectal, liver, endometrial, breast, pancreas, thyroid, uterus, or lung cancer or melanoma.

12. The method according to any one of claims 8 to 11, further comprising selecting the subject for a clinical trial for the treatment if and only if mitotic activity and / or cell proliferation is detected, or excluding the subject from the clinical trial if and only if mitotic activity and / or cell proliferation is not detected.

13. The method according to any one of claims 8 to 11, further comprising selecting the subject for the treatment if and only if mitotic activity and / or cell proliferation is detected, or excluding the subject from the treatment if and only mitotic activity and / or cell proliferation is not detected.

14. The method according to any one of claims 8 to 13, wherein detecting the presence or absence of mitotic activity comprises contacting the sample with a compound that binds MPM-2 or phospho Histone H3 (PHH3), or with haematoxylin and eosin.180539633.

115. The method according to any one of claims 8 to 14, wherein detecting the presence or absence of cell proliferation comprises contacting the sample with a compound that binds Ki-67, proliferating cell nuclear antigen (PCNA), Ki-S2, K1-S5, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MCM10, CAF-1 p60, CAF-1 pl50, NAV2, Unc-53, CDC6, CDC7, CDC7 protein kinase, Dbf4, CDC14, CDC14 protein phosphatase, CDC45, topoisomerase 2 alpha, DNA polymerase delta, replication protein A (RPA), replication factor C (RFC) or FEN1.

16. The method of claim 14 or claim 15, wherein the compound is an antibody.

17. The method according to any one of claims 8 to 13, wherein detecting the presence or absence of cell proliferation comprises determining a transcript level of Ki-67, proliferating cell nuclear antigen (PCNA), Ki-S2, Ki-S5, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, MCM10, CAF-1 p60, CAF-1 pl50, NAV2, Unc-53, CDC6, CDC7, CDC7 protein kinase, Dbf4, CDC14, CDC14 protein phosphatase, CDC45, topoisomerase 2 alpha, DNA polymerase delta, replication protein A (RPA), replication factor C (RFC) or FEN1.

18. The method according to any one of claims 1 to 17, further comprising administering the treatment to the subject or having the treatment administered to the subject.

19. The method according to any one of claims 1 to 18, wherein the treatment is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, and viro therapy.

20. The method of claim 19, wherein the treatment comprises immunotherapy.

21. The method of claim 20, wherein the immunotherapy comprises administering a checkpoint inhibitor.

22. The method of claim 21, wherein the checkpoint inhibitor inhibits PD-1, CTLA- 4, PD-L1 or any combination thereof.180539633.

123. The method according to any one of claims 1 to 22, wherein the treatment induces programmed cell death.

24. The method according to any one of claims 1 to 6, wherein the treatment induces cytotoxicity through RIPK3 or procaspase 8 effector pathways.

25. The method according to any one of claims 1 to 24, wherein the treatment comprises administering a therapeutically effective amount of a hedgehog signalling pathway inhibitor.

26. The method according to any one of claims 1 to 25, wherein the treatment comprises administering a therapeutically effective of a recombinant DNA virus comprising an expression cassette encoding interferon gamma, and operable for expression of the encoded interferon gamma in mammalian cancer cells.

27. The method according to any one of claims 1 to 26, wherein the treatment comprises administering a therapeutically effective amount of a hedgehog signalling pathway inhibitor and a recombinant DNA virus comprising an expression cassette encoding interferon gamma, and operable for expression of the encoded interferon gamma in mammalian cancer cells.

28. The method of claim 26 or claim 27, wherein the treatment comprises intralesional administration of the recombinant DNA virus.

29. The method according to any one of claims 1 to 28. wherein the subject to be treated is a human subject.

30. The method according to any one of claims 1 to 29, further comprising obtaining the sample from the subject prior to the detection step.

31. A method for stratifying or having stratified a group of human subjects for a clinical trial of a candidate treatment for basal cell carcinoma, the method comprising detecting the presence or absence of adnexal differentiation and / or adnexal involvement in a skin lesion sample from a subject in the group of human subjects, and selecting the subject for the clinical trial if no adnexal differentiation180539633.1and / or adnexal involvement is detected, or excluding the subject from the clinical trial if adnexal differentiation and / or adnexal involvement is detected.

32. A method for treating or having treated basal cell carcinoma in a subject, the method comprising administering a therapeutic agent to the subject, wherein the subject to be treated was determined to have a likelihood of a response to the therapeutic agent using a method according to any one of claims 1 to 30.

33. Use of a therapeutic agent in the manufacture of a medicament for treating basal cell carcinoma in a subject determined to have a likelihood of a response to the therapeutic agent using a method according to any one of claims 1 to 30.

34. A therapeutic agent for use in treatment of basal cell carcinoma in a subject determined to have a likelihood of a response to the therapeutic agent using a method according to any one of claims 1 to 30.

35. A method for stratifying or having stratified a group of human subjects for a clinical trial of a candidate treatment for cancer, the method comprising detecting the presence or absence of mitotic activity and / or cell proliferation in a sample from a subject in the group of human subjects, and selecting the subject for the clinical trial if mitotic activity and / or cell proliferation is detected, or excluding the subject from the clinical trial if no mitotic activity and / or cell proliferation is not detected.

36. A method for treating or having treated cancer in a subject, the method comprising administering a therapeutic agent to the subject, wherein the subject to be treated was determined to have a likelihood of a response to the therapeutic agent using a method according to any one of claims 8 to 28.

37. Use of a therapeutic agent in the manufacture of a medicament for treating cancer in a subject determined to have a likelihood of a response to the therapeutic agent using a method according to any one of claims 8 to 28.

38. A therapeutic agent for use in treatment of cancer in a subject determined to have a likelihood of a response to the therapeutic agent using a method according to any one of claims 8 to 28.180539633.

139. A method for excluding analysis of a response outcome in a clinical trial for treatment of basal cell carcinoma in a subject, the method comprising, following the treatment, detecting the presence or absence of adnexal differentiation and / or adnexal involvement in a skin lesion sample from the subject, and providing a result indicating inclusion of the response outcome for the analysis if no adnexal differentiation and / or adnexal involvement is detected, or providing a result indicating exclusion of the response outcome if adnexal differentiation and / or adnexal involvement is detected.180539633.1

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