Preventative treatment of HRD associated cancer

Abstract

The present invention relates to the treatment of homologous recombination deficiency associated cancer by prophylactic administration of an immune checkpoint inhibitor, and associated methods.

Description

[0001] Preventative Treatment of HRD Associated Cancer

[0002] Field of Invention

[0003] The present invention relates to the treatment of homologous recombination deficiency associated cancer by prophylactic administration of an immune checkpoint inhibitor.

[0004] Background

[0005] Despite major advances in the development of immune and targeted-therapies, the molecular and cellular complexity of late-stage tumours remain a major barrier to developing a 'silver bullet' cancer cure. This makes a strong case for focusing on identifying those at risk of developing cancer and intervening prophylactically to prevent tumour development. One of the major hurdles for the early detection of premalignant disease is our poor understanding of tumour initiating events. Historically, cancer research has focused on the identification of putative cancer driver genetic alterations and mutations. However, more recent studies found cancer driver mutations in healthy tissues suggesting that mutational events are not the sole drivers of tumour initiation. These findings highlight how little is understood about the impact of such genetic alterations on the cellular dynamics of the tissue, including changes in the immune and stromal microenvironment during the early stages of transformation. By identifying the early cellular and molecular changes that occur in the tissue, we could develop effective early detection and intervention strategies.

[0006] There is therefore a need to develop early interventions for the preventative treatment of cancer in high-risk subjects.

[0007] Summary of the Invention

[0008] One of the barriers for breast cancer prevention and treatment is our poor understanding of the dynamic cellular shifts that naturally occur within the breast and how these changes contribute to tumour initiation. The present inventors have used single cell RNA sequencing (scRNA-seq) to compile a Human Breast Cell Atlas (HBCA) assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mammoplasties. The data from more than 800,000 cells identified 41 cell subclusters distributed across the epithelial, immune, and stromal compartments. The inventors found that the contribution of these different clusters varied according to the natural history of the tissue. Factors that are known to modulate the risk of developing breast cancer such as age, parity, and germline mutation affected the homeostatic cellular state of the breast in different ways; however, none of the changes observed were restricted to a single cell type. Surprisingly the inventors also found that immune cells from BRCA1 / 2 carriers had prominent expression of immune checkpoint inhibitors and a distinct gene expression signature indicative of potential immune exhaustion which was validated by immunohistochemistry. This suggests that immune escape mechanisms could manifest in non- cancerous tissues during very early stages of tumour initiation.

[0009] These early signs of immune cell inhibition observed in tissues of BRAC1 and BRAC2 mutation carriers, suggests that immune escape mechanisms might manifest very early during tumour initiation. Based on this finding the present inventors are investigating current immunotherapy drugs as an early intervention to prevent HRD associated cancer development, in individuals at risk of developing a HRD associated cancer, particularly individuals with BRCA1 and BRCA2 mutations.

[0010] An aspect of the invention relates to an immune checkpoint inhibitor for use in a method for the preventative treatment of a HRD associated cancer in a subject wherein the method comprises prophylactically administering one or more immune checkpoint inhibitor to said subject.

[0011] An aspect of the invention relates to a method for the preventative treatment of a HRD associated cancer in a subject wherein the method comprises prophylactically administering an immune checkpoint inhibitor to said subject.

[0012] An aspect of the invention relates to a method of identifying a subject at risk of developing a HRD associated cancer comprising: screening a biological sample, obtained from a subject, for one or more cell comprising an immune exhausted phenotype.

[0013] An aspect of the invention relates to a method of identifying a subject at risk of developing a HRD associated cancer comprising: screening a biological sample, obtained from a subject, for one or more luminal adaptive secretory precursor cell (LASP) comprising an aberrant phenotype and / or increased expression of Csn2.

[0014] An aspect of the invention relates to a method of identifying a subject as suitable for prophylactic treatment with one or more immune checkpoint inhibitor comprising: selecting a subject, screening a biological sample obtained from said subject for the presence of an immune exhausted phenotype, and selecting said subject for prophylactic treatment with one or more immune checkpoint inhibitor based on the presence of the immune exhausted phenotype. An aspect of the invention relates to a method of identifying a subject as suitable for prophylactic treatment with one or more immune checkpoint inhibitor comprising: selecting a subject, screening a biological sample obtained from said subject for the presence of one or more LASP comprising an aberrant phenotype and / or one or more LASP comprising increased expression of Csn2, and selecting said subject for prophylactic treatment with one or more immune checkpoint inhibitor.

[0015] An aspect of the invention relates to a method of therapy monitoring, in a subject at risk of developing a HRD associated cancer, wherein the therapy comprises prophylactic treatment with one or more immune checkpoint inhibitor comprising: selecting a subject who has received or is receiving prophylactic treatment with one or more immune checkpoint inhibitor, screening a biological sample obtained from said subject for the presence of one or more LASP comprising an aberrant phenotype and / or the expression level of Csn2 present on one or more LASP, wherein response to therapy is indicated by a reduction in the level of LASP comprising an aberrant phenotype and / or a decrease in expression of Csn2 on one or more LASP.

[0016] Figures

[0017] Figure 1. Overview of the Human Breast Cell Atlas. A schematic highlighting the overall experimental design and the cell types we aimed to capture in the Atlas. The diagram highlights the overall number of donors sequenced and how they are distributed among the various subgroups alongside the number of Donor FFPEs that have been analysed by multiplex immunostaining. The global uniform manifold approximation and projection (UMAP) representation of the nal dataset is coloured by general cell types captured from all 161 samples processed as part of this Atlas.

[0018] Figure 2. Major cellular groups identified in the Human Breast Cell Atlas, (a) Uniform manifold approximation and projection (UMAP) plots of the epithelial, stromal and immune cell compartments coloured and labelled by subcluster annotations. Doublets / stripped nuclei are plotted in grey, (b) Dot plots summarising the known markers used to identify cell types and a selection of genes distinguishing each subcluster for the epithelial (left), stromal (middle) and immune (right) compartments. Each column corresponds to a specific cell subcluster and the rows correspond to a list of key marker genes (expression normalised per gene), brackets on the right side of each plot detail the cell type or subcluster that these genes mark.

[0019] Figure 3. Age and parity affect the homeostatic cellular state of the breast, (a) Milo cell neighbourhood differential abundance plots of the significant (FDR < 0.05) changes in the breast composition with age, blocking for the effects of parity (mammoplasty donors; n=22). We test the effects of age as a continuous scale ranging from 19-65 years, with the colour gradient scale representing log fold changes per year. Blue represents enrichment with age whilst red denotes depletion with age. (b) Beeswarm plot of the log fold changes in the Milo neighbourhoods with age, grouped into each cell type subcluster. Neighbourhoods with a significant change in cellular abundance are coloured as indicated. Log fold changes are per year due to the continuous age scale tested, (c) Milo cellular neighbourhood differential abundance plots of the significant (FDR < 0.05) changes in the breast composition with parity (i.e. nulliparous versus parous), blocking for the effects of ageing (mammoplasty donors; n=22). Blue represents enrichment with parity whilst red denotes depletion with parity, (d) Beeswarm plot of the log fold changes of the Milo neighbourhoods with parity, grouped into each cell type subcluster. Neighbourhoods with a significant change in cellular abundance are coloured as indicated.

[0020] Figure 4. High-risk BRCA1 (HR-BR1) and BRCA2 (HR-BR2) germline mutations associated with increased proportions of immune cells in the breast, (a) Summary plot of average Milo differential abundance results comparing the cellular composition of the breast from average risk (AR) donors against HR-BR1 and HR-BR2 donors, blocking for the effects of both age and parity. To enable fair comparisons, neighbourhoods were computed to be shared by AR / HR-BR1 and AR / HR-BR2 tests. The plot summarises the mean and variance of log fold changes for each cell subcluster in both the HR-BR1 and HR-BR2 cohorts. A zero log fold change represents the same subcluster proportions as AR donor tissue on average, whilst positive (negative) log fold changes denote subcluster enrichment (depletion) in the relative HR donor breast, (b) Example of whole section output using the Ultivue staining technology. DAPI, pan-Cytokeratin (panCK), CD8, CD4, CD3, CD68, PDL1 , PD1 and FOXP3 staining with colours indicated in -gure are shown on a whole section and magni-ed insets from a representative donor. Scale bars represent 100 pm in the insets, 2 mm in the whole section image, (c) Ultivue staining showing panCK (white), CD8 (green), CD4 (red) and DAPI (blue) of breast sections from representative AR (n=10), HR-BR1 (n=11) and HR-BR2 (n=8) donors as indicated. Scale bars represent 100 pm.

[0021] Figure 5. High-risk BRCA1 (HR-BR1) and BRCA2 (HR-BR2) donor breast tissue display increased expression of immune checkpoint inhibition and exhaustion markers, (a) Boxplots visualising the change in mean expression (log-transformed counts) for immune checkpoint ligand PDL1 , identified as significantly upregulated in HR-BR1 (LASP, LHS and Macrophage) and HR-BR2 (LASP and Macrophage) donors with respect to average risk (AR) controls, (b) Top, Ultivue staining showing pan-Cytokeratin (panCK, white), PDL1 (orange), PD1 (green) and DAPI (blue) of a breast section from an HR donor showing an example of epithelial expression of PDL1 . Bottom, Ultivue staining showing panCK (white), PDL1 (orange), CD68 (green) and DAPI (blue) of a breast section from an HR donor showing examples of CD68 cells expressing PDL1 . Scale bars represent 100 pm. (c) Dot plot displaying the expression of several key immune checkpoint receptors expression in a range of cytotoxic lymphoid subclusters comparing between AR, HR-BR1 and HR-BR2 donor groups. Expression is normalised per gene, (d) Top, immunofluorescence staining showing panCK (white), HAVCR2 (red) and DAPI (blue) of representative breast sections from AR and HR donors. Bottom, immunofluorescence staining showing panCK (white), TIGIT (red) and DAPI (blue) of a representative breast section from AR and HR donors. Arrowheads point at examples of positive cells. Each staining is representative of two (AR, HR-BR2) or three (HR-BR1) donor samples. Scale bars represent 100 pm. (e) Bar plot showing the percentage of PD1 + / CD3+ double positive cells located in non- epithelial areas (ie. not intercalated with the epithelium) in HR-BR1 / 2 donor compared to AR donors in whole Ultivue slides (see methods). The p-values are calculated with non-parametric Wilcox tests.

[0022] Figure 6. The integrated Human Breast Cell Atlas (iHBCA) provides quantitative comparison of cell type annotations across seven of the largest single cell RNA sequencing datasets for the breast, (a) A schematic showing the curation of the iHBCA combining seven of the largest single cell RNA sequencing datasets for the breast. The diagram highlights the composition and sample heterogeneity captured by the iHBCA. The central plot shows a global uniform manifold approximation and projection (UMAP) representation of the dataset coloured by transferred subcluster annotations (see Fig 2) from the Human Breast Cell Atlas (HBCA). Annotation labels were mapped using CellTypist logistic regression models (see methods), (b) A set of six confusion matrices showing the cell type / subcluster comparisons between each of the published datasets and our own subcluster annotations. Each cell (row A, column B) shows the percentage of cells of type A in the original dataset that are mapped to cell type B in our HBCA subcluster annotations. Note: LC1 / 2 cells from the Twigger dataset are cells thought to only appear in the lactating gland and are hence absent from the HBCA cohort causing their nonsensical logistic regression mapping.

[0023] Figure 7. Quantification of Ultivue and Immunofluorescence images, (a) Bar plots showing the ratio of CD4+ (left) and CD4+ / PD1 + double-positive (right) cells to PanCK+ cells in whole Ultivue tissue slides from average risk (AR) and high-risk BRCA1 / 2 (HR-BR1 / 2) donors, (b) Bar plots showing the ratio of CD8+ (left) and CD8+ / PD1 + double-positive (right) cells to PanCK+ cells in whole Ultivue tissue slides from average risk (AR) and high-risk BRCA1 / 2 (HR-BR1 / 2) donors. The p-values are calculated with non-parametric Wilcox test with * indicating p < 0.05. (c) Bar plots showing the percentage of TIGIT+ (left), HAVCR2+ (middle) and GZMH+ (right) cells from the immuno-fluorescence images (n=2 for AR and HR-BR2, n=3 for HR-BR1) shown in Figure 5. (d) Bar plot showing the percentage of CD3+ cells located in non-epithelial areas (i.e., not intercalated with the epithelium) in HR-BR1 / 2 donor compared to AR donors in whole Ultivue slides (see methods), (e) Example segmentation and calling of positive cells of an immunofluorescence (GZMH) image.

[0024] Figure 8. Anti-PD-1 treatment in Brcal mouse model causes reduction in aberrant LASP and Csn2 expression, (a) Stacked bar plot showing the proportion of LASP cells of the canonical and aberrant subtype in both control (n=3) and PD1 inhibitor treated (n=3) mice, (b) Box and whisker plot of the average Csn2 expression in LASP cells from both control (n=3) and PD1 -inhibitor treated (n=3) mice.

[0025] Detailed Description of the Invention

[0026] The embodiments of the invention will now be further described. In the following passages, different embodiments are described. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.

[0027] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).

[0028] The nomenclatures used in connection with, and the laboratory procedures and techniques of, biochemistry, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Suitable assays to measure the properties as set out above are also described in the examples.

[0029] The present invention relates to prophylactic treatment with immune checkpoint inhibitors in order to prevent or delay the development or progression of a HRD associated cancer. As such an aspect of the invention relates to an immune checkpoint inhibitor for use in a method for the preventative treatment of a HRD associated cancer in a subject wherein the method comprises prophylactically administering one or more immune checkpoint inhibitor to said subject.

[0030] An aspect of the invention relates to method for the preventative treatment of a HRD associated cancer in a subject wherein the method comprises prophylactically administering an immune checkpoint inhibitor to said subject.

[0031] An aspect of the invention relates to the use of an immune checkpoint inhibitor in the manufacture of a medicament for the preventative treatment of a HRD associated cancer in a subject wherein the immune checkpoint inhibitor is prophylactically administered to said subject. The homologous recombination (HR) repair pathway is a highly accurate DNA repair pathway that allows the repair of DNA double stranded breaks. Multiple mediators are involved in the HR pathway, notably BRCA1 and BRCA2. Alterations in many of the genes involved in HR have been identified as prevalent in various cancers. Alterations in the genes involved in HR can lead to a HR deficient phenotype wherein the cell comprises a defective HR repair mechanism. The term “homologous recombination deficiency associated cancer” or“HRD associated cancer” has its general meaning in the art and refers to cancer displaying defective homologous recombination (HRD)-mediated DNA repair which causes genomic instability and hyperdependence on alternative DNA repair mechanisms for survival. A HRD associated cancer may comprise a mutation, alteration or aberration within one or more of the HR associated genes. The one or more mutation, alteration or aberration may be present in one or more of the following genes BRCA1 , BRCA2, 53BP1 , ATM, ATR, ATRIP, BARD1 , BLM, BRIP1 , DMC1 , MRE11A, NBN, PALB2, RAD50, RAD51 , RAD51 B, RAD51 C, RAD51 D, RIF1 , RMI1 , RMI2, RPA1 , TOP3A, TOPBP1 , XRCC2, and XRCC3. The one or more mutation in a HR associated gene may comprise a germline mutation, and / or a somatic mutation. The one or more alteration or aberration in a HR associated gene may include structural modifications, deletions, histone modifications such as histone acetylation, and / or DNA modifications such as DNA methylation. The term “HRD associated cancer” includes but is not limited to BRCA-associated cancer. In some embodiments of the invention described herein the HRD associated cancer refers to breast cancer, ovarian cancer, prostate cancer, fallopian tube cancer, primary peritoneal cancer, stomach cancer, colorectal cancer, gall bladder cancer, cervical cancer, pancreatic cancer, lung cancer, head and neck cancer and melanoma, wherein the cancer comprises a HR deficient phenotype. In an embodiment the HRD associated cancer refers to breast cancer wherein the cancer comprises a HR deficient phenotype.

[0032] In an embodiment the preventative treatment is performed in a subject that is at risk of developing a HRD associated cancer. A subject at risk of developing a HRD associated cancer may comprise a DNA pathogenic event which increases the risk of HRD associated cancer. In an embodiment the DNA pathogenic event that increase the risk of HRD associated cancer may comprise one or more mutation, alteration or aberration. The one or more mutation that increases the likelihood of developing a HRD associated cancer may be a germline mutation and / or a somatic mutation. The one or more alteration or aberration that increases the likelihood of developing a HRD associated cancer may include DNA structural modifications, deletions, histone modifications such as histone acetylation, and / or DNA modifications such as DNA methylation. In certain embodiments the subject at risk of developing a HRD cancer may comprise a BRCA pathogenic variant. In some embodiments the one or more mutation that increases the likelihood of developing a HRD cancer may be a BRCA pathogenic variant. In an embodiment the HRD associated cancer may be a BRCA associated cancer.

[0033] An aspect of the invention relates to an immune checkpoint inhibitor for use in a method for the preventative treatment of a BRCA associated cancer in a subject wherein the method comprises prophylactically administering one or more immune checkpoint inhibitor to said subject. A further aspect of the invention relates to method for the preventative treatment of a BRCA associated cancer in a subject wherein the method comprises prophylactically administering an immune checkpoint inhibitor to said subject. A further aspect of the invention relates to the use of an immune checkpoint inhibitor in the manufacture of a medicament for the preventative treatment of a BRCA associated cancer in a subject wherein the immune checkpoint inhibitor is prophylactically administered to said subject. In an embodiment the subject comprises a DNA pathogenic event which increases the risk of BRCA associated cancer for example the subject comprises a BRCA pathogenic variant.

[0034] The term “BRCA-associated cancer” as used herein has its general meaning in the art and refers to cancer associated with BRCA mutation or BRCA expression deficiency. The term “BRCA- associated cancer” refers to cancer selected from but not limited to cancer associated with BRCA1 and / or BRCA2 mutation, in particular inactivation of BRCA1 and / or BRCA2 genes, cancer associated with BRCA1 and / or BRCA2 expression deficiency, homologous recombination deficiency (HRD) cancer and / or BRCA-deficiency cancer, such as basal-like, luminal, and HER2-overexpressing carcinomas, breast, ovarian, and prostate tumours harbouring BRCA1 / 2 mutations and other cancers. In some embodiments, the term “BRCA- associated cancer” refers to breast cancer, ovarian cancer, prostate cancer, fallopian tube cancer, primary peritoneal cancer, stomach cancer, colorectal cancer, gall bladder cancer, cervical cancer, pancreatic cancer, lung cancer, head and neck cancer and melanoma with BRCA1 and / or BRCA2 mutation or BRCA1 and / or BRCA2 expression deficiency. The term “BRCA-associated cancer” also refers to chemo-resistance BRCA-associated cancer such as PARP inhibitor (PARPi) resistant BRCA-associated cancer, PARPi-resistant HRD tumours including tumours with somatic reversion of BRCA 1 / 2 mutation and subsequent HR restoration, cisplatin resistant BRCA-associated cancer and cisplatin-resistant BRCA1 and BRCA2-mutated tumours including tumours with somatic reversion of BRCA1 / 2 mutation and subsequent HR restoration.

[0035] The term “BRCA1 / 2” refers to BRCA1 and / or BRCA2, more particularly BRCA1 and BRCA2.

[0036] In an embodiment the HRD associated cancer and / or the BRCA associated cancer may be selected from breast cancer, ovarian cancer, prostate cancer, fallopian tube cancer, primary peritoneal cancer, stomach cancer, colorectal cancer, gall bladder cancer, cervical cancer, pancreatic cancer, lung cancer, head and neck cancer or melanoma. The BRCA associated cancer may comprise a BRCA pathogenic variant. The term “BRCA pathogenic variant” may be used interchangeably with “BRCA pathological variant”. As used herein the term BRCA pathogenic variant refers to a BRCA gene comprising a mutation, genetic alteration or genetic aberration that increases the risk of developing cancer. The mutation or aberration may be present in BRCA1 and / or BRCA2. In an embodiment the BRCA pathogenic variant comprises one or more mutation in BRCA1 and / or BRCA2. The BRCA pathogenic variant may comprise a germline mutation and / or a somatic mutation. The BRCA pathogenic variant may comprises a mutation that causes loss of function of BRCA1 and / or BRCA2. The BRCA pathogenic variant may comprise a genetic alteration or aberration including but not limited to DNA structural modifications, deletions, histone modifications such as histone acetylation, and / or DNA modifications such as DNA methylation.

[0037] The mutations, genetic alterations and / or genetic aberrations described herein may be identified using any standard technique in the art. In an embodiment the mutations, genetic alterations and / or genetic aberrations are identified via sequencing. Sequencing may be performed using whole genome, whole exome, targeted exome, transcriptome, and methylome sequencing. Techniques are known in the art for comparing sequences to identify the presence of mutations, for example sequence alignment may be used.

[0038] The present invention relates to the prophylactic use of immune checkpoint inhibitors in the preventative treatment of a HRD associated cancer and / or a BRCA associated cancer. The prophylactic use of immune checkpoint inhibitors may prevent or delay development of a HRD associated cancer and / or a BRCA associated cancer. As used herein, the term "preventative treatment" refers to prophylactic or preventive treatment, including treatment of subjects at risk of developing the disease or suspected to be in the initial stages of developing the disease. As such prophylactic or preventative treatment may be administered to subjects that are at risk or at high risk of developing the disease or in the early stages of the disease wherein it is not yet detectable. Subjects may be identified as at risk or at high risk of developing a HRD associated cancer based on genetic screening or familial history of cancer. Prophylactic administration refers to administration of said treatment in order to prevent or delay development of a disease. Prophylactic administration of said treatment may be performed prior to the subject developing detectable disease. Prophylactic administration of said treatment may be performed by administering said treatment to a subject at risk of developing the disease or suspected to be in the initial stages of developing the disease.

[0039] The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0040] As used herein, the term "effective amount" means an amount of the immune checkpoint inhibitor as described herein, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired preventative or prophylactic effect under the conditions of administration.

[0041] The preventative treatment of the present invention comprises the administration of one or more immune checkpoint inhibitor. Immune checkpoint inhibitors are molecules which have significant promise in the treatment of cancer and are useful to treat a variety of types of cancers. As will be appreciated by those in the art, checkpoint inhibitors are used to increase the immune response but are not generally tumour specific in their action, as such they generally inhibit the suppression of the immune system, generally leading to T cell activation, which in turn leads to greater immune response to cancerous cells and thus treatment.

[0042] According to the invention the checkpoint inhibitor may be selected from a PD-1 inhibitor, a PD- L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, a T cell immunoglobulin and ITIM domain (TIGIT) inhibitor, a V-domain Ig suppressor of T cell activation (VISTA) inhibitor, a BTLA inhibitor, a T-cell immunoglobulin and mucin domain-3 (TIM-3) inhibitor, a PD-L2 inhibitor, B7-H3 inhibitor, B7-H4 inhibitor, CD160 inhibitor, CD244 inhibitor, KLRG1 inhibitor, B7-1 inhibitor, B7-2 inhibitor, a KIR inhibitor, Galectin-9 inhibitor, NECTIN2 inhibitor, NECTIN3 inhibitor, an adenosine pathway inhibitor or an IDO inhibitor. In a preferred embodiment the checkpoint inhibitor may be selected from a PD-1 inhibitor and / or a PD-L1 inhibitor. In some embodiments the checkpoint inhibitor disrupts the PD-1 PD-L1 interaction.

[0043] In an embodiment the checkpoint inhibitor may be selected from a small molecule inhibitor or an antibody or antigen binding fragment thereof.

[0044] A PD-1 inhibitor refers to a compound or molecule that inhibits programmed cell death protein 1 , examples of suitable PD-1 inhibitors include but are not limited to pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, INCMGA00012 (MGA012), AMP-224, AMP-514, acrixolimab. A PD-L1 inhibitor refers to a compound or molecule that inhibits programmed death ligand 1 , examples of suitable PD-L1 inhibitors include but are not limited to atezolizumab, avelumab, durvalumab, KN035, cosibelimab, AUNP12, CA-170, BMS-986189. A CTLA-4 inhibitor refers to a compound or molecule that inhibits cytoctoxic T-lymphocyte associated protein 4 also known as CD152. Examples of suitable CTLA-4 inhibitors include but are not limited to ipilimumab and tremelimumab. A LAG-3 inhibitor refers to a compound or molecule that inhibits Lymphocyteactivation gene 3, examples of suitable LAG-3 inhibitors include but are not limited to relatlimab.

[0045] A TIGIT inhibitor refers to a compound or molecule that inhibits T cell immunoglobulin and ITIM domain (TIGIT), examples of TIGIT inhibitors include but are not limited to tiragolumab, domvanalimab, vibostolimab, ociperlimab, tiragolumab. A VISTA inhibitor refers to a compound or molecule that inhibits V-domain Ig suppressor of T cell activation, examples of VISTA inhibitors include but are not limited to CA-170. A BTLA inhibitor refers to a compound or molecule that inhibits B- and T-lymphocyte attenuator A TIM-3 inhibitor refers to a compound or molecule that inhibits T-cell immunoglobulin and mucin domain-3 (TIM-3) inhibitor, A PD-L2 inhibitor refers to a compound or molecule that inhibits programmed death ligand-2. A B7-H3 inhibitor refers to a compound or molecule that inhibits B7 Homolog 3 also known as CD276. A B7-H4 inhibitor refers to a compound or molecule that inhibits B7 Homolog 4 also known as VTCN1 . A CD160 inhibitor refers to a compound or molecule that inhibits cluster of differentiation 160. A CD244 inhibitor refers to a compound or molecule that inhibits cluster of differentiation 244. A KLRG1 inhibitor refers to a compound or molecule that inhibits killer cell lectin-like receptor subfamily G member 1 .A B7-1 inhibitor refers to a compound or molecule that inhibits B7-1 also known as CD80. A B7-2 inhibitor refers to a compound or molecule that inhibits B7-2 also known as CD86. A KIR inhibitor refers to a compound or molecule that inhibits one or more killer-cell immunoglobulin-like receptor, the compound or molecule may also inhibit one or more of the KIR genes. A Galectin-9 inhibitor refers to a compound or molecule that inhibits Galectin- 9. A NECTIN2 inhibitor refers to a compound or molecule that inhibits Nectin Cell Adhesion Molecule 2. A NECTIN3 inhibitor refers to a compound or molecule that inhibits Nectin Cell Adhesion Molecule 3. An adenosine pathway inhibitor refers to a compound or molecule that inhibits one of the proteins in the adenosine pathway such as A2AR and / or A2BR. An IDO inhibitor refers to a compound or molecule that inhibits indoleamine 2,3-dioxygenase, examples of suitable IDO inhibitors include but are not limited to epacadostat (INCB24360), navoximod (GDC-0919), and linrodostat (BMS-986205).

[0046] Various combinations of checkpoint inhibitors may be used according to the invention for example combining two or more checkpoint inhibitors. In an embodiment the checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a VISTA inhibitor, a BTLA inhibitor, a TIM-3 inhibitor, a PD-L2 inhibitor, B7-H3 inhibitor, B7-H4 inhibitor, CD160 inhibitor, CD244 inhibitor, KLRG1 inhibitor, B7-1 inhibitor, B7-2 inhibitor, a KIR inhibitor, Galectin-9 inhibitor, NECTIN2 inhibitor, NECTIN3 inhibitor, an adenosine pathway inhibitor or an IDO inhibitor, or a combination thereof.

[0047] In certain embodiments of the present invention combinations of immune checkpoint inhibitors are used. Where a combination of immune checkpoint inhibitors is used the combination may result in an additive effect, or a more than additive effect for example a synergistic effect. A combination therapy is defined as affording an “additive effect”, “synergistic effect” or a “synergistic treatment” if the effect is therapeutically superior, as measured by, for example, the extent of the response (e.g. apoptosis or cell viability), the response rate, the time to disease progression or the survival period, to that achievable on dosing one or other of the components of the combination therapy at its conventional dose. For example, the effect of the combination of immune checkpoint inhibitors is additive if the effect is therapeutically superior to the effect achievable with said checkpoint inhibitors alone. For example, the effect of the combination treatment may be synergistic if the effect of the combination treatment is greater than the effect of the individual treatments added together.

[0048] For example a PD-1 inhibitor may be combined with one or more of a PD-L1 inhibitor, a CTLA- 4 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a VISTA inhibitor, a BTLA inhibitor, a T-cell immunoglobulin and mucin domain-3 (TIM-3) inhibitor, a PD-L2 inhibitor, B7-H3 inhibitor, B7-H4 inhibitor, CD160 inhibitor, CD244 inhibitor, KLRG1 inhibitor, B7-1 inhibitor, B7-2 inhibitor, a KIR inhibitor, Galectin-9 inhibitor, NECTIN2 inhibitor, NECTIN3 inhibitor, an adenosine pathway inhibitor or an IDO inhibitor. A PD-L1 inhibitor may be combined with one or more of a PD-1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a VISTA inhibitor, a BTLA inhibitor, a T-cell immunoglobulin and mucin domain-3 (TIM-3) inhibitor, a PD-L2 inhibitor, B7- H3 inhibitor, B7-H4 inhibitor, CD160 inhibitor, CD244 inhibitor, KLRG1 inhibitor, B7-1 inhibitor, B7-2 inhibitor, a KIR inhibitor, Galectin-9 inhibitor, NECTIN2 inhibitor, NECTIN3 inhibitor, an adenosine pathway inhibitor or an IDO inhibitor. A PD-L2 inhibitor may be combined with one or more of a PD-1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a VISTA inhibitor, a BTLA inhibitor, a TIM-3 inhibitor, a PD-L1 inhibitor, B7-H3 inhibitor, B7-H4 inhibitor, CD160 inhibitor, CD244 inhibitor, KLRG1 inhibitor, B7-1 inhibitor, B7-2 inhibitor, a KIR inhibitor, Galectin-9 inhibitor, NECTIN2 inhibitor, NECTIN3 inhibitor, an adenosine pathway inhibitor or an IDO inhibitor.

[0049] The combinations of checkpoint inhibitors may comprise 2, 3, 4, 5 checkpoint inhibitors in combination.

[0050] In an embodiment the immune checkpoint inhibitor is selected from a PD-L1 inhibitor, a PD-L2 inhibitor, a PD-1 inhibitor or a combination thereof. In an embodiment the immune checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 or the PD-1 / PD-L2 pathway. In an embodiment the immune checkpoint inhibitor is selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, INCMGA00012 (MGA012), AMP-224, AMP-514, acrixolimab, atezolizumab, avelumab, durvalumab, KN035, cosibelimab, AUNP12, CA-170, BMS-986189 or a biosimilar thereof. The present inventors have previously shown that luminal adaptive secretory precursor cells. (LASPs), also referred to as luminal progenitor cells, play a key role in cancer development in BRCA carriers. As such in an embodiment the HRD associated cancer may be characterised by the presence of LASPs. The LASPs may comprise canonical LASPs marked by strong expression of typical LASP marker including but not limited to Aldh1 a3, Kit, Cd14, Fcgbp and Lurapl l. The LASPs may comprise aberrant LASPs marked by additional expression of several markers not traditionally expressed in LASP cells, including but not limited to Csn2, Csn1s1 , Elf5, Lalba, Cdknl a, Cdkn2a, Trp63, Krt5, Col3a1 , Col1 a1. The LASPs may comprise a combination of canonical and aberrant LASP. The LASPs may express a biomarker such as Csn2, Csn1s1 , Elf5, Lalba, Cdkn2a and any other differentially expressed gene used to define the aberrant LASP population of cells.

[0051] In an embodiment the presence of a HRD associated cancer or the risk of a subject developing a HRD associated cancer may be assessed by a suitable assay. The present inventors have shown that changes in LASPs are indicative of HRD associated cancers (Bach et al., 2021). As such the presence or risk of developing a HRD associated cancer may be assessed by determining the LASP status of a subject, for example in terms of the level of aberrant LASPs and / or the level of Csn2 expression on LASPs. The assay may comprise quantifying the level of aberrant LASPs in a subject. The assay may comprise quantifying the expression of Csn2 in a subject. The assay may comprise quantifying the expression of Csn2 present on LASPs. The quantification of canonical and aberrant LASPs and / or expression of Csn2 may be assessed by RNA sequencing. The RNA sequencing may include, but is not limited to, single-cell RNA sequencing, differential gene expression, direct RNA sequencing, small RNA sequencing, messenger RNA sequencing or next-generation sequencing. Where the level of aberrant LASPs is increased compared to a reference value this may be indicative of a subject at risk of developing a HRD associated cancer. Where the expression level of Csn2 present on LASPs is increased compared to a reference value this may be indicative of a subject at risk of developing a HRD associated cancer.

[0052] The identification of the LASP status of a subject may be performed by screening a sample from said subject. This screening step may be performed as part of the method of the present invention, or it may be performed separately, and this information obtained for example via medical records.

[0053] In an embodiment the subject is at risk of developing a HRD associated cancer. In an embodiment the subject comprises a BRCA pathogenic variant. The subject may be further characterised as comprising an immune exhausted phenotype. In an embodiment a subject comprising an immune exhausted phenotype may be at high risk of developing a HRD associated cancer. The subject may further comprise one or more cell which presents an immune exhausted phenotype. The term “immune exhausted phenotype” refers to the markers that may be identified in lymphocytes that have been exposed to chronic stimulation. Key indicators of immune exhaustion include but are not limited to loss of T cell function, upregulation of immune checkpoint molecules, attenuated effector cytotoxicity, reduced cytokine production.

[0054] The identification of the immune exhausted phenotype may be performed by screening a sample from said subject. This screening step may be performed as part of the method of the present invention, or it may be performed separately, and this information obtained for example via medical records.

[0055] The methods of the present invention may comprise a step of screening a subject to identify if they are at high risk of developing a HRD associated cancer, this screening step may involve identifying markers of immune exhaustion or aberrant LASP status. In an embodiment the method for the preventative treatment of a HRD associated cancer further comprises; screening a biological sample from said subject for one or more cell comprising an immune exhausted phenotype, for one or more LASP comprising an aberrant phenotype, and / or for the expression level of Csn2 present on one or more LASP.

[0056] The biological sample may be selected from blood, plasma, serum, sputum, bile, tears, urine and / or tissue. The biological sample may be obtained via a tissue biopsy and / or a liquid biopsy. A tissue sample may be obtained using standard methods for example tissue biopsy. In some embodiments the tissue sample is a breast tissue sample. In an embodiment the biological sample is a liquid biopsy sample. The liquid biopsy may comprise blood, serum and / or plasma.

[0057] In an embodiment the biological sample is fresh or frozen.

[0058] The biological sample may be obtained from a subject who needs to be assessed for the risk of developing a HRD associated cancer. The subject may be identified, for example due to familial history of cancer or mutational status such as having a BRCA mutation or expression deficiency.

[0059] The immune exhausted phenotype may be identified or characterised by an increased expression of PD-L1 , PD1 , CTLA4, LAG3, TIGIT, TIM3, VISTA, BTLA, TIM-3, PD-L2, B7-H3, B7-H4, CD160, CD244, KLRG1 , B7-1 , B7-2, KIR, Galectin-9, NECTIN2, NECTIN3, IDO or a combination thereof.

[0060] The immune exhausted phenotype may be present on CD8 T cell and / or a CD4 T cell. The one or more cell comprising an immune exhausted phenotype may be selected from a CD8 T cell, a CD4 T cell, an NK cell and / or an NKT cell. The immune exhausted phenotype may be present on a CD8 TEM cell, a CD8 TCI cell, an NKT cell and / or an NK cell. The one or more cell comprising an immune exhausted phenotype may be selected from a CD8 TEM cell, a CD8 TCI cell, an NKT cell and / or an NK cell.

[0061] The immune exhausted phenotype may be further characterised by increased PD-L1 expression on various immune cells. In certain embodiments the increased PD-L1 expression is present on LASPs, macrophages and / or LHS. The term “LASP” refers to luminal adaptive secretory precursor cells. The term “LHS” refers to luminal hormone sensing cells.

[0062] In some embodiments the immune exhausted phenotype is further characterised by an increased level of CD8 T cells, optionally CD8 TCI cells.

[0063] In embodiments that refer to identifying an increased level this may be identified by comparing the level of expression to a reference value. Said reference value may be obtained from a healthy individual for example an individual who does not have cancer or a high risk of developing cancer. Said reference value may be obtained from an individual who does not comprise a HRD associated pathogenic variant e.g., an individual who does not comprise a mutation alteration or genetic aberration in one or more HR gene which increases their risk of HRD associated cancer. Said reference value may be obtained from an individual who does not comprise a BRCA pathogenic variant, e.g., an individual who does not comprise a mutation or genetic aberration in BRCA1 or BRCA2 which increases their risk of BRCA associated cancer.

[0064] In an embodiment the one or more cell comprising an immune exhausted phenotype are localised to a non-epithelial area. The term “non-epithelial area” refers to one or more region comprising limited or no detectable expression of one or more epithelial marker. Epithelial markers may be selected from EPCAM, KRT14, KRT8, and / or pan cytokeratin. In an embodiment a non-epithelial area may refer to a region with no detectable expression of one or more epithelial markers selected from one or more of EPCAM, KRT14, KRT8 and pan- cytokeratin. In an embodiment, where a breast tissue sample is screened, the one or more cells comprising an immune exhausted phenotype are localised to a non-epithelial area within the breast tissue.

[0065] In an embodiment the one or more cell comprising an immune exhausted phenotype localised to non-epithelial areas, is characterised PD1 , CTLA4, LAG3, TIGIT, TIM3, VISTA, BTLA, TIM- 3, B7-H3, B7-H4, CD160, CD244, KLRG1 , B7-1 , B7-2, KIR, Galectin-9, NECTIN2, NECTIN3, and / or IDO expression. In an embodiment the one or more cell comprising an immune exhausted phenotype localised to non-epithelial areas, is characterised PD1 expression. In an embodiment the immune exhausted phenotype is further characterised by PDL1 expression on CD8 T cells and / or NKT cells, optionally CD8 TEM cells and / or CD8 TCI cells. In an embodiment the immune exhausted phenotype is further characterised by LAG3 and / or TIGIT expression on CD8 T cells and / or NKT cells, optionally CD8 TEM cells and / or CD8 TCI cells. In an embodiment the immune exhausted phenotype is further characterised by expression of TIM-3 on NKT cells. In any of the aforementioned embodiments the expression of the immune checkpoint molecule may be increased compared to a reference value.

[0066] In an embodiment the one or more cell comprising an immune exhausted phenotype localised to non-epithelial areas, is characterised PD1 + / CD3+ expression.

[0067] The immune checkpoint inhibitor may be administered at any suitable dose. For example, the immune checkpoint inhibitor is administered at a dose sufficient to restore cells to a non-immune exhausted phenotype. The skilled person would be able to determine that the cells had been restored to a non-immune exhausted phenotype using techniques standard in the art for example identifying a reduction in one or more of the immune checkpoint molecules identifying an increase in T cell function, identifying an increase in effector cytotoxicity, identifying an increase in cytokine production.

[0068] The amount of the therapeutic that is effective / active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0069] In an embodiment the immune checkpoint inhibitor is administered at a dose in the range of 0.1 ug / kg to 250 mg / kg, 0.1 ug / kg to 100 mg / kg, 0.1 ug / kg to 50 mg / kg, 0.1 ug / kg to 10 mg / kg, 0.2 ug / kg to 10 mg / kg, 0.5 ug / kg to 10 mg / kg, 0.7 ug / kg to 10 mg / kg, 1 .0 ug / kg to 10 mg / kg, 1.5 ug / kg to 10 mg / kg, 2.0 ug / kg to 10 mg / kg, 5.0 ug / kg to 10 mg / kg, 10 ug / kg to 10 mg / kg, 20 ug / kg to 10 mg / kg, 50 ug / kg to 10 mg / kg, 75 ug / kg to 10 mg / kg.

[0070] In an embodiment administration of the one or more checkpoint inhibitor is performed on a daily, weekly, monthly basis. In an embodiment administration of the or more checkpoint inhibitor is performed on a bi-weekly, bi-monthly basis. The immune checkpoint inhibitor may be provided as a pharmaceutical composition e.g., comprising a checkpoint inhibitor as described herein and optionally a pharmaceutically acceptable carrier. The immune checkpoint inhibitor or pharmaceutical composition may be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation. In some embodiments the immune checkpoint inhibitor is administered by intravenous infusion, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravesical, intradermal, topical or subcutaneous administration. The checkpoint inhibitor may be administered by any convenient route. In some embodiments the checkpoint inhibitor is administered by intravenous infusion, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravesical, intradermal, topical or subcutaneous administration. Wherein one or more checkpoint inhibitors are provided as separate compositions they may be administered by the same or different routes and may be formulated the same or differently as appropriate.

[0071] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally.

[0072] In certain embodiments one or more further preventative treatment of a HRD associated cancer and / or a BRCA associated cancer is administered. The further preventative treatment may be selected from any suitable therapy for example surgery or small molecule therapy.

[0073] In embodiments where the HRD associated cancer and / or BRCA associated cancer is breast cancer the further preventative treatment of breast cancer may comprise a mastectomy. In embodiment the preventative small molecule therapy comprises tamoxifen, anastrozole, and / or raloxifene.

[0074] An aspect of the invention related to a method of identifying a subject at risk of developing a HRD associated cancer comprising; screening a biological sample, obtained from a subject, for one or more cell comprising an immune exhausted phenotype.

[0075] An aspect of the invention related to a method of identifying a subject at risk of developing a HRD associated cancer comprising; screening a biological sample, obtained from a subject, for one or more LASP comprising an aberrant phenotype and / or increased expression of Csn2. According to the method of wherein the method of identifying a subject at risk of developing a HRD associated cancer, the biological sample may be selected from blood, plasma, serum, sputum, bile and / or tissue. In an embodiment the biological sample is a tissue sample. A tissue sample may be obtained using standard methods for example tissue biopsy. In some embodiments the tissue sample is a breast tissue sample. In an embodiment the biological sample is a liquid biopsy sample. The liquid biopsy may comprise blood, serum and / or plasma.

[0076] In an embodiment the biological sample is fresh or frozen.

[0077] In an embodiment the method of identifying a subject at risk of developing a HRD associated cancer may comprise screening a biological sample, obtained from a subject, for one or more cell comprising an immune exhausted phenotype, and / or screening a biological sample, obtained from said subject, for one or more LASP comprising an aberrant phenotype and / or increased expression of Csn2.

[0078] The immune exhausted phenotype may be characterised by any of the features as described above. In particular the immune phenotype may be identified by screening the biological sample for an increased expression of one or more of the following markers: PD-L1 , PD1 , CTLA4, LAG3, TIGIT, TIM3 VISTA, BTLA, TIM-3, PD-L2, B7-H3, B7-H4, CD160, CD244, KLRG1 , B7-1 , B7- 2, KIR, Galectin-9, NECTIN2, NECTIN3, and / or IDO expression.

[0079] The one or more cell comprising said immune exhausted phenotype may be a lymphocyte. In an embodiment the cell comprising said immune exhausted phenotype is selected from a CD8 T cell and / or a CD4 T cell. In an embodiment the one or more cell comprising an immune exhausted phenotype is selected from a CD8 TEM cell, a CD8 TCI cell, an NKT cell and / or an NK cell.

[0080] The method of identifying a subject at risk of developing a HRD associated cancer may further comprise a step of screening said biological sample for an increased level of CD8 T cells, optionally CD8 TCI cells.

[0081] The method of identifying a subject at risk of developing a HRD associated cancer may further comprise screening said biological sample for one or more cells comprising an immune exhausted phenotype localised in non-epithelial areas within the biological sample.

[0082] In certain embodiments said biological sample is screened for PD1 + / CD3+ double positive cells localised in non-epithelial areas. In certain embodiments the HRD associated cancer and BRCA pathogenic variant may comprises any of the further features set out above.

[0083] In certain embodiments the biological sample is screened for one or more LASPs comprising aberrant phenotype. The aberrant phenotype may be characterised by one or more markers including but not limited to Csn2, Csn1s1 , Elf5, Lalba, Cdknl a, Cdkn2a, Trp63, Krt5, Col3a1 , Coll a1 , as such the step of screening ma involve screening for expression level of one or more of the aforementioned markers.

[0084] In certain embodiments the biological sample is screened for one ore more LASP comprising increased expression of the marker Csn2. The level of expression of Csn2 may be increased compared to a reference value.

[0085] In certain embodiments the method of identifying a subject at risk of developing a HRD associated cancer may comprise characterising the LASPs present in said subject. For example, quantification of canonical and / or aberrant LASPs may be performed, and / or the expression level of Csn2 present on said LASPs may be quantified. The quantification of canonical and aberrant LASP and / or expression of Csn2 may be assessed by RNA sequencing. The presence of aberrant LASPs and / or overexpression of Csn2 may indicate the subject is at risk of developing a HRD associated cancer.

[0086] Once an subject has been identified as having one or more of an immune exhausted phenotype, increased aberrant LASPs, or increased expression of Csn2 they may be selected for preventative treatment with one or more immune checkpoint inhibitor as set out herein. The immune checkpoint inhibitor may be selected from any of those disclosed herein. In an embodiment the immune checkpoint inhibitor is selected from a PD-L1 inhibitor, a PD-L2 inhibitor, a PD-1 inhibitor or a combination thereof.

[0087] An aspect of the invention relates to a method of identifying a subject as suitable for prophylactic treatment with one or more immune checkpoint inhibitor comprising: selecting a subject, screening a biological sample obtained from said subject for the presence of an immune exhausted phenotype, selecting said subject for prophylactic treatment with one or more immune checkpoint inhibitor based on the presence of the immune exhausted phenotype.

[0088] An aspect of the invention relates to a method of identifying a subject as suitable for prophylactic treatment with one or more immune checkpoint inhibitor comprising: selecting a subject, screening a biological sample obtained from said subject for the presence of one or more LASP comprising an aberrant phenotype and / or one or more LASP comprising increased expression of Csn2, and selecting said subject for prophylactic treatment with one or more immune checkpoint inhibitor.

[0089] The methods of identifying a subject as suitable for prophylactic treatment with one or more immune checkpoint inhibitor, may comprise any of the additional features as set our herein above.

[0090] In the methods of the present invention the subject may be at risk of developing a HRD associated cancer. The subject may comprise any of the further features set out herein above. For example the subject may comprises one or more mutation alteration or aberration in one or more HR associated gene. The subject may be at risk of developing a BRCA associated cancer. The subject may comprise a BRCA pathogenic variant. The BRCA pathogenic variant may comprise one or more mutation alteration or aberration in BRCA1 and / or BRCA2. A HRD associated cancer may comprise a mutation, alteration or aberration within one or more of the HR associated genes. The one or more mutation, alteration or aberration may be present in one or more of the following genes BRCA1 , BRCA2, 53BP1 , ATM, ATR, ATRIP, BARD1 , BLM, BRIP1 , DMC1 , MRE1 1A, NBN, PALB2, RAD50, RAD51 , RAD51 B, RAD51 C, RAD51 D, RIF1 , RMI1 , RMI2, RPA1 , TOP3A, TOPBP1 , XRCC2, and XRCC3. The HRD associated cancer may comprise one or more mutation in a HR associated gene such as a germline mutation, and / or a somatic mutation. The HRD associated cancer may comprise one or more alteration or aberration in a HR associated gene including but not limited to structural modifications, deletions, histone modifications such as histone acetylation, and / or DNA modifications such as DNA methylation. The HRD associated cancers include but are not limited to BRCA-associated cancer. In some embodiments the HRD associated cancer refers to breast cancer, ovarian cancer, prostate cancer, fallopian tube cancer, primary peritoneal cancer, stomach cancer, colorectal cancer, gall bladder cancer, cervical cancer, pancreatic cancer, lung cancer, head and neck cancer and melanoma, wherein the cancer comprises a HR deficient phenotype.

[0091] In an embodiment the subject is selected for prophylactic treatment with one or more immune checkpoint inhibitor based on the presence of one or more LASP comprising an aberrant phenotype and / or one or more LASP comprising increased expression of Csn2.

[0092] The method of identifying a subject as suitable for prophylactic treatment with one or more immune checkpoint inhibitor, may comprise screening a biological sample for the presence of an immune exhausted phenotype, the presence of one or more LASP comprising an aberrant phenotype and / or one or more LASP comprising increased expression of Csn2. As such any of the steps related to screening for an immune exhausted phenotype may be combined with the steps related to identifying the presence of one or more LASP comprising an aberrant phenotype and / or one or more LASP comprising increased expression of Csn2.

[0093] As shown in the Examples prophylactic treatment with an immune checkpoint inhibitor in a BRCA mouse model resulted in a reduction in aberrant LASPs and Csn2 expression i.e. two key indicators of cancer development. As such response to treatment may be monitored via these markers. An aspect of the invention relates to a method of therapy monitoring, in a subject at risk of developing a HRD associated cancer, wherein the therapy comprises prophylactic treatment with one or more immune checkpoint inhibitor comprising: selecting a subject who has received or is receiving prophylactic treatment with one or more immune checkpoint inhibitor, screening a biological sample obtained from said subject for the presence of one or more LASP comprising an aberrant phenotype and / or expression level of Csn2 on one or more LASP, wherein a response to therapy is indicated by a reduction in the level of LASP comprising an aberrant phenotype and / or a decrease in expression of Csn2 on one or more LASP.

[0094] The step of screening a biological sample may be performed at one or more time points over the course of treatment, in order to determine changes in the level of aberrant LASP and / or expression level of Csn2. As such the level of aberrant LASP and / or expression of Csn2 may be compared to a reference value to determine changes over time. Said reference value may be obtained from the subject prior to beginning treatment. Said reference value may be obtained from a healthy individual for example an individual who does not have cancer or a high risk of developing cancer. Said reference value may be obtained from an individual who does not comprise a HRD associated pathogenic variant e.g., an individual who does not comprise a mutation alteration or genetic aberration in one or more HR gene which increases their risk of HRD associated cancer. Said reference value may be obtained from an individual who does not comprise a BRCA pathogenic variant, e.g., an individual who does not comprise a mutation or genetic aberration in BRCA1 or BRCA2 which increases their risk of BRCA associated cancer.

[0095] In an embodiment the subject has received or is receiving prophylactic treatment with one or more immune checkpoint inhibitor, wherein the one or more immune checkpoint inhibitor is selected from any one of those described herein. In an embodiment the immune checkpoint inhibitor is selected from a PD-L1 inhibitor, a PD-L2 inhibitor, a PD-1 inhibitor or a combination thereof.

[0096] The methods of the present invention may be performed in vivo, in vitro and / or ex vivo. In certain embodiments wherein the method relates to identifying subjects at risk of developing a HRD associated and / or BRCA associated cancer or identifying subjects as suitable for treatment these methods may be performed in vitro or ex vivo.

[0097] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications.

[0098] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0099] The term “comprising” or “comprises” where used herein means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of’ or “consists essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components and the like.

[0100] The term “consisting of’ or “consists of’ means including the components specified but excluding other components.

[0101] Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists essentially of’ or “consisting essentially of’, and also may also be taken to include the meaning “consists of’ or “consisting of’.

[0102] The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention, as set out herein are also applicable to all other aspects or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each aspect or exemplary embodiment of the invention as interchangeable and combinable between different aspects and exemplary embodiments.

[0103] It should be understood that while the use of words such as “preferable”, “preferably”, “preferred” or “more preferred” in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as “a,” “an,” or “at least one,” are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

[0104] EXAMPLES

[0105] Introduction

[0106] One of the biggest challenges in treating BRCA associated cancers, such as breast cancer, is the heterogeneous nature of the disease. We have limited understanding of how early divergences from the homeostatic breast subtypes lead to tumour heterogeneity. Whilst large- scale cancer genomic studies indicate that different breast cancer subtypes are enriched for certain mutations, not all phenotype variability and tumour behaviour can be explained by mutations alone. Studies in mice have shown that the cell-of-origin may contribute to the phenotype of the resulting tumour. Defining which cell type leads to which kind of tumour is complicated by a growing list of environmental and epidemiological risk factors, such as age, which not only affect overall incidence, but also outcome. Such risk factors can themselves be modulated by other factors; for example, the age-dependent risk in breast cancer is greatly reduced by pregnancy early in life, whereas predisposing germline mutations (e.g. BRCA1 / 2) greatly increases age-associated risk. How these risk factors interact and impact tissue homeostasis remains to be fully understood. To do this, it is crucial to characterise cell types and cellular states present under different physiological conditions.

[0107] To tackle this problem, studies in the mouse have leveraged single cell genomics (i.e. scRNA- seq) to determine the gene expression profile of individual mammary epithelial cells across embryonic and adult developmental stages. Similar to the mouse, after birth the human mammary gland continues to develop. At birth the human breast consists of a ductal structure with well-defined terminal ductal lobular units (TDLUs) similar to those found in the adult. Prior to puberty, the breast grows in proportion to the rest of the body. The onset of puberty triggers the expansion of the TDLU to form adult lobules and to fill the surrounding stroma. Consisting of the vasculature system, fibroblasts and immune cells, the stromal compartment remains severely understudied. Despite this, mounting evidence suggests that changes in the microenvironment are a major contributing factor in tumorigenesis and an important target for novel therapeutics. Together, these motivate the study of the complex interactions between epithelial cells and the surrounding stromal. Recent single cell transcriptomic and proteomic studies of human reduction mammoplasty and tumour samples have begun to catalogue the various epithelial compartments in the breast. However, larger sample sizes, encompassing major developmental changes and risk-modulating factors, are needed to generate a comprehensive transcriptomic map of all adult breast cell subtypes and states. To this end, we report the use of scRNA-seq to compile a comprehensive Human Breast Cell Atlas (HBCA) of over 800,000 cells collected from 55 women across various adult development stages.

[0108] Example 1 Identification of a suitable tissue cohort

[0109] Here we present a comprehensive human breast cell atlas, enabling us to map how cellular composition changes as a function of various biological and environmental factors. To assist study of a wide range of these factors we identified a cohort of healthy breast tissue samples from the Breast Cancer Now Tissue bank that had (up to 80) health and lifestyle records available (Fig 1). Our cohort consisted of tissue samples from 22 women who had undergone reduction mammoplasties, 27 women carrying a BRCA1 or BRCA2 mutation or other family histories (who had risk reduction prophylactic mastectomies) and 6 contralateral mastectomies from BRCA1 carriers that had breast cancer in one breast and had the second breast removed to reduce the risk of further tumours (Fig 1). The samples had a wide distribution of values across the various risk modifiers such as age, parity status and menopause. Additionally, we collected a range of metadata on further risk modifying factors that may prove beneficial to future studies. All samples were pre-processed by the tissue bank to isolate the epithelial and stromal / immune enriched compartments (Fig 1). These were then processed to single cell level and viable cells were loaded on a 10x chromium chip for single cell capturing to enable scRNA-seq. For the epithelial enriched compartment, we also used fluorescence-activated cell sorting (FACS), as previously described, to enrich for luminal progenitor cells (EPCAM+ , CD49f + ), which have been proposed to be the cell of origin for some breast cancers.

[0110] Following sequencing, more than one million cells were identified. After several quality control and computational doublet calling steps, 801 ,360 cells were taken forward for downstream analysis (Fig 1). To combine our 45 separate sequencing batches we used scVI22 to produce a batch corrected embedding for use in downstream analysis. More details on the sample preparation and analysis can be found in the methods section. Coarse cell type annotation revealed that we sequenced over 350,000 epithelial cells, and 400,000 stromal and immune cells (Fig 1).

[0111] Example 2 Major cell subtypes identified in the Human Breast Cell Atlas

[0112] Within the epithelial compartment we used canonical lineage markers to identify three major cell types: the luminal adaptive secretory precursor (LASP; also known as Luminal Progenitor,), luminal hormone sensing (LHS), and basal-myoepithelial (BMYO) (Fig 2). Iterative clustering identified several subclusters within each cell type based on unique gene expression profiles. The majority of these subclusters were found in all 55 donors, albeit at varying proportions. Of note, we found no distinct LASP cluster strongly marked by milk-biosynthesis genes (CSN2 / 3, LALBA; Fig 2b). Instead, we find most LASP heterogeneity is defined by proportions of standard marker gene expression. LASP1 / 2 / 3 show high expression of LASP specific markers (ALDH1 A3, SLPI) in contrast to LASP4. Additionally, LASP2 is notable for its co-expression of both LASP (ALDH1A3) and BMYO markers (KRT14, KRT5) (Fig 2b). We also identified LASP5 as a small population of proliferating cells marked by the canonical proliferation marker MKI67 and mitosis related genes (AURKB, TOP2A; Fig 2b). Similarly, within the LHS compartment, upregulation of AREG marked LHS1 cells, while upregulation of oestrogen and progesterone receptors (ESR1 and PGR) marked LHS2 cells. Subcluster LHS3 shows an expression pattern distinguished by high SERPINA1 and PIP expression alongside increased expression of some LASP markers (ALDH1A3, SLPI; Fig 2b). The two BMYO subclusters are distinguished by expression of canonical BMYO markers KRT5 and KRT14 (high in BMYO1) as well as OXTR (high in BMYO2; Fig 2b).

[0113] Within the stromal compartment we identified four fibroblast (FB), four vascular endothelial cell (VE), two lymphatic endothelial cell (LE) and five perivascular (PV) subclusters, which were present across all donors (Fig 2). FB1 shows increased expression of genes related to extracellular matrix (ECM) formation (DCN, LUM, COL1A2), whereas FB2 is marked by several genes related to ECM disassembly (MMP2, MMP3, MMP10 MMP12 and SH3PXD2B). We found FB3 to generally possess a quite distinctive transcriptional profile compared to the other FB subclusters and was marked by high CLU and GREM1 expression (Fig 2b). The venous, arterial, capillary and angiogenic tip endothelial subclusters were identified using a range of previously described marker genes23-25. The LE compartment, distinguished by canonical markers CCL21 and PDPN, splits into two groups that can be separated by their expression of chemokines (CXCL1 and CXCL8) and some angiogenic tip cell markers (ANGPT2, PXDN) both of which are highly expressed in LE1 , but lowly expressed in LE2. The perivascular subclusters appear in two main groups, with PV1 / 2 having low expression of genes relating to muscular functions (ACTA2, TAGLN2) and pericyte markers (RGS5) 26 which are high in PV3 / 4 / 5. Using previously described immune cell markers we were also able to identify a variety of lymphoid and myeloid cell types including five T cell subtypes, NKT cells, NK cells, ILC cells, three B cell subtypes, plasma cells, dendritic cells and two macrophage subclusters (Fig 2). To provide robust 100-gene signatures for each identified subcluster, we used pseudobulk one-versus-all differential expression testing to identify an extensive list of marker genes. These lists show high specificity to our cell type and subcluster annotations and offer many potentially novel cell type markers for use of the community. We found all major cell types represented in all donors regardless of BRCA, age or parity status.

[0114] Our sub-clustering also identified two Donor Derived Clusters (DDC), each consisting primarily of cells from just one donor (Donor 17 and Donor 37 respectively, Fig 2a). Both DDC1 and DDC2 show high expression of LASP marker genes (ALDH1A3 and SLPI but not KIT, Fig 2b). Despite this, they also show a mix of other epithelial lineage markers from LHS and BMYO cell types. Additionally, DDC1 shows particularly high expression of MMP3, as well as PIP and MUCL1 , which were otherwise predominantly expressed in LHS3 within the epithelium (Fig 2b). Despite the expression of mixed lineage markers, both DDC1 and DDC2 were not associated with batch, have low doublet scores and typical quality control metric distributions. We carried out an additional quality control step to distinguish between single nuclei (stripped nuclei) from single cells using gene signatures described in a previous study. This identified several such stripped nuclei clusters (coloured grey in UMAPs), however, these did not overlap with the DDCs, suggesting that the DDCs are indeed single cells. Together, this supports the DDC clusters as genuine and distinct donor specific cells.

[0115] Due to the mixed marker expression and rare patient specific nature of the clusters we then investigated the possibility these cells show early signs of transformation. To investigate the similarity with known tumour signatures, we looked at the four breast cancer subtype gene scores across our epithelial subclusters. Both DDC clusters stood out from the remaining epithelial subclusters showing elevated gene scores for HER2+ and basal-like subtypes. To explore this further, we used inferCNV31 which compares RNA expression binned across the genome between two groups of cells to predict copy number variations (CNVs) at a single cell level. To maximise robustness against cell type and donor specific expression patterns across the genome, we used a reference set covering a range of epithelial, stromal and immune cells sampled from different mammoplasty donors alongside the non-epithelial cells of the DDC donors. The results of this analysis identified prominent CNV profiles in both DDC1 and DDC2 subclusters. This analysis identified a range of deletions in chromosomes 3 and 10, duplications in chromosomes 1 , 1 1 , 12 and 17 as well as evidence of chromosome 19 duplication. Many of these aberrations were shared between DDC1 and DDC2. We additionally found a range of predicted duplications that appeared subcluster specific with DDC2 showing distinctive duplications on chromosomes 17 (containing ERRB2) and 22. When comparing the profiles of DDC1 and DDC2 to previously published CNV profiles of the four tumour subtypes, particularly Luminal A and TNBC, we observe several similarities, including the amplifications observed in chromosome 1 and duplication of chromosome 19.

[0116] Example 3 Changes in healthy breast composition under different physiological conditions

[0117] We first consider how natural factors such as age and parity impact the composition of the breast. To avoid confounding our results with changes resulting from differences in BRCA status, we exclusively consider the 22 reduction mammoplasty donors. To gain high-resolution insight into cellular differential abundance and compositional shifts, we used Milo differential abundance testing. This approach first groups cells into small overlapping neighbourhoods based on similar gene expression (methods). Then, in each neighbourhood, we look for statistically significant enrichment of cells from each condition tested using flexible generalised linear models, thereby avoiding both statistical confounding and the inherent biases placed by fine cell type clustering. We found that most of the significant changes driven by age occur within the epithelial compartment. In the LASP compartment, older samples display an enrichment of LASP2 / 4 cells, while regions of the LASP1 / 3 subclusters are enriched in younger individuals (Fig 3a, b). The LASP clusters enriched in older women have reduced expression of traditional LASP markers, consistent with previous studies showing increased LASP lineage infidelity with age. In the LHS compartment, we found an enrichment most prominently in the LHS2 and to a lesser extent LHS3 subclusters with age, while in the BMYO compartment we found a significant decrease in the proportion of BMYO1 cells (Fig 3a, b). Outside the epithelial compartment, there are few significant differences with age except for an enrichment in a subset of FB1 cells and the FB3 subcluster, as well as depletion of plasma cells (Fig 3a, b).

[0118] In contrast, parity has a more widespread impact on the cellular composition of the epithelial, stromal and immune compartments (Fig 3c, d) which could reflect the largescale tissue remodelling of the breast that occurs during pregnancy. In the LASP compartment, there is a decreased proportion of LASP4 cells as a function of parity, despite enrichment for the LASP1 / 2 / 3 subclusters in parous women. In the LHS and BMYO compartments, there is an enrichment of LHS3 and BMYO1 subclusters in parous donors. We note that many of the changes in cellular abundance seen within the epithelial compartment are in the opposite direction when considering age or parity as the covariate, which could contribute to contrasting breast cancer risk posed by each. We see some evidence of this with overall increased proportions of plasma, CD8 TEM and CD4 T cell types in the profiled parous donors. In the stromal compartment, our findings suggest a transition from ACTA2 expressing PV3 / 4 / 5 towards PV1 / 2-type perivascular cells as a function of parity, as well as increases in proportions of VEA and VEAT cells and an overall increase in most FB subclusters excluding FB3 (Fig 3c, d).

[0119] Example 4 Impact of high-risk BRCA1 and BRCA2 germline mutations on breast cellular composition and transcriptional profile

[0120] To determine mammary cell state shifts in high risk (HR) donors compared with average risk (AR) donors, we analysed cell profiles from reduction mammoplasty donors (n=22) and donors with BRCA1 (n=11) and BRCA2 (n=11) germline mutations (denoted HR-BR1 and HR-BR2 respectively). First, we tested for differential expression between AR and HR-BR1 as well as between AR and HR-BR2 cohorts, accounting for the effects of both age and parity. Despite similar statistical power, we noted that HR-BR1 epithelial cells have more significant transcriptional changes than the respective HR-BR2 cells. This is particularly clear in the BMYO compartment which showed only one (HLA-DQB1) significantly upregulated gene in HR-BR2 cells compared to the AR cells. Of note in the LASP compartment, we see evidence for the upregulation of milk-biosynthesis (CSN2 / 3, CSN1 S1 and LALBA) in HR-BR1 LASP cells particularly. The upregulation of milk-biosynthesis genes is similarly interesting to tumour development, with recent mouse studies proposing these genes as possible markers of pre- tumour progression. These results suggest a similar process may also characterise early signs of malignant progression in the human breast. Unsurprisingly, the expression of milkbiosynthesis genes also has a strong relationship to parity, with a strong upregulation in parous donors regardless of BRCA status.

[0121] To explore other cellular differences, particularly in the immune and stromal compartment, we use Milo to compare AR reduction mammoplasty donors against HR-BR1 or HR-BR2 donors and identify compositional changes that may contribute to the higher risk of breast cancer development. To allow easy comparisons between HR-BR1 and HR-BR2 induced changes we summarised this in a ’Milo signature’ plot averaging neighbourhood log fold changes per donor for each subcluster (Fig 4a). Both HR-BR1 and HR-BR2 donors (though more prominent in the latter) show shifts towards the VEAT and LE1 populations (Fig 4a). However, one of the most significant changes in the HR cohorts is the large increase in the proportion of lymphocytes, particularly CD4, CD8 and NK / NKT cells (Fig 4a). The most prominent changes are a two-fold enrichment in the HR-BR1 donors of CD8 TC1 cells, which are characterised by high IFNG and TNF expression - both of which are known to be pro-inflammatory (Fig 2b and Fig 4a). To confirm this observation we obtained matched slides from 30 of the sequenced donors to perform orthogonal immunofluorescence validation (see methods for details). This allowed us to assess up to eight markers simultaneously on tissue sections from AR, HR-BR1 and HR-BR2 donors (Fig 4b). In agreement with Milo, we observe evidence of increased proportions of CD4, CD8 and NK / NKT positive cells in HR-BR1 and HR-BR2 donors within regions of the tissue (Fig 4c).

[0122] A possible impact of this immune response was hinted to in the differential gene expression analysis of the three epithelial compartments. Further exploration of these results revealed increased expression of PDL1 (also known as CD274) in HR-BR1 / 2 LASPs and Macrophages as well as HR-BR1 LHS cells relative to AR controls (Fig 5a). Despite PDL1 expression being rare, we were able to identify PDL1 + epithelial and CD68+ cells in multiple HR donors using immunofluorescence (Fig 5b). This observation led us to explore more immune checkpoint inhibitors and markers of immune cell exhaustion in our cohort, including PDCD1 (also known as PD1), CTLA4, LAG3, TIGIT and HAVCR2 (also known as TIM3). We see general increases in the frequency and level of expression across immune checkpoint / exhaustion genes in both HR-BR1 and HR-BR2 donors (Fig 5c). There are modest increases in the expression of PDCD1 (PDL1 receptor) itself in the CD8 TEM, CD8 TC1 and NKT (HR-BR1 specific) subclusters. We also see increased expression of LAG3 and TIGIT in the CD8 TC1 / TEM and NK / NKT cell types of HR-BR1 / 2 donors alongside increased HAVCR2 expression in NKT cells from HR donors (Fig 5c). This is supported by immunofluorescence analysis showing evidence of increased exhaustion markers in matched HR donor tissue slides compared to AR controls (Fig 5d and Fig 7). Spatially, we found that immune cells marked by PD1 expression were preferentially localised outside the epithelium in HR-BR1 and HR-BR2 donors compared to AR counterparts (Fig 5e). Collectively, these results suggest that whilst there is increased immune activity in the HR donors, the immune system is showing signs of exhaustion and suppressed function in these tissues.

[0123] The Milo analysis also revealed several differences between the HR-BR1 and HRBR2 donors. This can be seen in subclusters of the epithelium, where the HR-BR1 donors showed strong enrichments for LASP4, LHS3 and BMYO2 cell subclusters not shared with HR-BR2 donors. Instead, the HR-BR2 donors showed decreased LASP4 and LHS2 subclusters in comparison to AR donors (Fig 4a).

[0124] Example 5 Comparison of cell type annotations across single cell breast studies

[0125] To facilitate the comparison of cell types and their subclusters defined within the HBCA cohort we integrated our data with six of the largest scRNA-seq studies of the healthy breast to form the first integrated Human Breast Cell Atlas (iHBCA). The iHBCA includes both fresh and frozen tissue prepared using a range of different protocols across multiple labs totalling 2.1 million cells from 286 individuals (Fig 6a). We used scVI to perform integration of the seven datasets to correct for any batch specific sequencing effects (see methods for more details) which preserved the general cell type structure from each of the seven studies. This analysis highlighted a major lack of consensus in the cell nomenclature used across datasets. To address this, we utilised a CellTypist logistic regression classifier (methods) which we trained based on the subcluster annotations of our HBCA cohort. Using this model, we were able to assign an identity to each individual cell from all datasets, using our HBCA annotation as the reference. The resulting mapped cell type annotations are visualised on the joint iHBCA UMAP (Fig 6a). To quantitatively summarise the label mapping efficiency and nomenclature comparisons we created confusion matrices showing the proportion of cells from each original dataset label mapped onto the corresponding HBCA cell type label (Fig 6b). Overall, this shows strong concordance of cell type annotations between datasets. In addition, we repeated the differential abundance testing on the iHBCA and confirmed many of the findings we reported earlier.

[0126] To facilitate the usefulness of the iHBCA as a community resource, we have curated pretrained CellTypist models based on the cell type annotations provided by each dataset (see data availability). These models will enable other researchers to easily map iHBCA labels onto their own datasets using minimal computational resources. This stands as a comprehensive resource to compare cell type and state annotations across different datasets.

[0127] Discussion

[0128] In this study, we describe a scRNA-seq Human Breast Cell Atlas generated by sequencing over 800,000 cells from 55 donors. The scale of this dataset has enabled us to delve into the entire breast composition, encompassing not only the epithelium but also the surrounding microenvironment. Due to the diversity of the samples, we were able to interrogate the data relative to several key breast cancer risk modifiers, such as age, parity and BRCA1 / 2 germline mutations, enabling us to uncover cellular interactions and compositional changes associated with each factor.

[0129] As expected, both age and parity affect the homeostatic cellular state of the breast. Although the changes observed are not restricted to any one type of cell, we could identify unique features for the two risk factors. In contrast to parity, which has a widespread impact on breast composition, the main changes associated with age are concentrated within the epithelial compartment. Most notably, we observed an enrichment of the LASP2 / 4 clusters, which are characterised by mixed lineage expression and reduced fidelity towards traditional LASP (luminal progenitor) markers. This is consistent with previous observations of increased LASP lineage infidelity with age15,30,34,35. The global impact of parity can be seen across the epithelial, stromal and immune compartments. Many of the effects observed in the epithelium oppose those observed with age such as strong changes in BMYO1 and LHS2 proportions alongside more subtle general shifts in the total proportion of LASP cells. Outside the epithelium, we note increased proportions of plasma, CD8 TEM and CD4 T cells in parous women (Fig 3). Overall, we found that the cellular composition changes observed with parity and age are complex and not restricted to any one type of cell. Thus, the impact of these changes needs to be considered collectively rather than in isolation given the contrasting impact of age and parity on breast cancer risk. It would be interesting to explore the impact of other factors that will likely influence the homeostatic gland in combination with age and parity such as hormonal status or menopause, which due to limited sample size and metadata availability couldn’t be assessed in our study.

[0130] Once age and parity are accounted for, we do not identify cell populations that are exclusively associated with BRCA1 / 2 germline mutation carriers, rather we observe shifts in the proportions of certain cell type subclusters. One of the most diverse cell populations we captured in this study is the LASP compartment, which is also the subpopulation of epithelial cells most associated with breast cancer21 . Some of the changes we observed here include decreased overall LASP proportions in both HR groups, with BRCA1 samples having an enrichment of the LASP4 population, similar to that seen with age. Additionally, there was strong enrichment for the LHS3 and BMYO2 subclusters in HR-BR1 donors that was not seen in the HR-BR2 donors (Fig 4a).

[0131] Some of the largest changes seen in our HR cohorts occurred in the immune compartment. In particular, we detected a significant immune expansion, prominently of the CD8 TC1 cells in HR- BR1 donors, accompanied by increased expression of canonical immune checkpoint / exhaustion receptors in both HR cohorts (Fig 4a and Fig 5). A similar phenotype was reported in the Fallopian tube of BRCA1 mutation carriers, suggesting commonalities across tissues. Interestingly, when considering transcriptional shifts in HR donors compared to AR donors, we observe an increase in PDL1 expression, mainly in LASPs and macrophages but also across some of the LHS cells (Fig 5a, b). Previous studies have shown that high expression of IFNG in CD8 T cells, resembling what we observe in the CD8 TC1 cell population, can directly drive PDL1 expression in melanoma38, suggesting that this expansion of CD8 TC1 cells could be contributing to the PDL1 induction in HR donors. The upregulation of PDL1 could be particularly interesting due to its key role in immune evasion and supporting a tumourigenic microenvironment. Given the observed increase in immune checkpoint / exhaustion markers in HR donor immune cells (Fig 5b, c) and the accumulating evidence of the involvement of LASPs in tumour initiation, our findings point towards an early epithelial immune-escape mechanism in the pre-malignant tissue driven by BRCA1 and BRCA2 germline mutations.

[0132] Within the stromal compartment, we found a strong enrichment of VEAT cells in HR donors, particularly HR-BR2 (Fig 5a). Predictive cell-cell interaction analysis (methods) suggests that SEMA3 and SEMA6 signalling pathways could be involved in mediating this enrichment. This agrees with a previous study where male BRCA mutation carriers were found to have increased proportions of endothelial progenitor cells. This warrants future investigations given the role of angiogenesis and vascular remodelling in tumourigenesis.

[0133] Finally, one of our primary objectives was to provide the community with a robust reference dataset that would enable seamless projection and integration with other datasets (methods). As an example of this potential, we produced the iHBCA which integrates seven of the largest scRNA-seq datasets, representing a variety of sample types and tissue processing approaches (Fig 6a). Overall, while we found strong concordance between cell types across datasets, the integration also revealed that the enrichment of the different cell types and subclusters varies greatly between datasets. We found that some of this variation could clearly be described by tissue storage and preparation differences between datasets. In particular, whether the cells were sequenced fresh following surgery or after collection from frozen tissue appeared to play a major role in the proportion of immune cell types and the transcriptional profile of the BMYO cells. We found that frozen tissue generally yielded more epithelial and stromal cells whilst fresh produced more immune cells. Another difference worth noting for future studies is the number of cells sampled per individual which varied greatly across the datasets. Our study sequences the largest number of cells per individual, which allowed us to perform high resolution differential abundance analysis through Milo as well as identify rare clusters in two of our donors (denoted DDC). Alongside mixed marker expression and increased tumour gene signatures, predictive CNV analysis suggested that these two clusters harboured genomic aberrations associated with early stages of tumourigenesis. Finally, the iHBCA presented here provides a framework for seamless integration of future sequencing datasets as we move towards larger cohorts that better represent the wider population.

[0134] Example 6 - prevention of tumour development in a Brcal mouse model

[0135] The present inventors have shown that the epithelial cell compartment that changed significantly in precancer was the aberrantly differentiated luminal cell. Our data revealed that immune cells also changed precancer and this was driven by the increase in cell-cell interaction with the newly formed aberrant epithelial cells. This is highlighted in our putative cell-cell interaction analysis which looks at the expression pattern of ligand-receptor signalling pathways across the various cell types within the mammary gland. This analysis revealed that indeed most of the changes in the receptor-ligand interactions were centred around the aberrantly differentiated cells and several immune cell types. The main immune cells affected during the premalignant stages are CD4 Treg cells and Macrophages. Treg cells are known to play a major role in attenuating the adaptive anti-tumorigenic immune response. There are several clinical trials investigating the impact of targeting Treg cells to improve immune surveillance and clearing of cancer cells. Equally, tumour associated macrophages play a role in promoting tumour growth and metastasis and there are several clinical trials looking into the disruption of these cells as cancer immunotherapy. However, there are currently no clinical trials investigating the disruption of tumour promoting immune cells in a chemo preventative setting. Based on our data there is a strong case for investigating such approach.

[0136] Therefore, in the present inventors aim to test adaptive immune checkpoint inhibitors which we found to be upregulated during aberrant differentiation (Figure 5). The inventors aim to prophylactically treat the Brcal mouse model with FDA approved (or near approved) drugs, including inhibitors which target checkpoint inhibitors (listed above).

[0137] We will investigate the impact of these compounds on the aberrant differentiation process and tumour development.

[0138] To test these agents in vivo we will use the Brcal mouse model which displays the aberrant differentiation phenotype around 30 weeks of age. To identify whether the immune checkpoint inhibitors affect aberrant differentiation at the cellular level, we will administer a single agent or its vehicle control at 30 weeks of age at a frequency of 1 dose every one or two weeks. The treatment for all cohorts will last for 5 weeks and mice will be monitored throughout the experiment for signs of tumour development. At the end of the 5 weeks, the mammary glands will be collected from at least 3 mice per cohort, and 3 corresponding vehicle control mice for scRNAseq analysis and the data will be compared to our published single cell genomics dataset9. In detail, from the mammary glands we will: i) store a sample for FFPE and histology, ii) store a sample for future protein analysis, iii) a sample will be used for scRNAseq to determine the presence of aberrant differentiated cells and any changes in the non-epithelial cells associated with treatments, iv) a sample will be used to analyse the mammary glands by FACS, to assess proportions and transitions within the epithelial and non-epithelial compartment.

[0139] The scRNAseq analysis of the mammary glands is essential as it will extensively determine if aberrant differentiation has been affected by the treatment and if there are any changes in the non-epithelial compartment. Assessing if the checkpoint inhibitors prevent the early immune and stromal changes associated with aberrant differentiation will also be crucial in the evaluation and potential of this strategy.

[0140] Preliminary results from this study have shown evidence suggestive of PD1 inhibitors efficacy in preventing signs of pre-cancer progression. After completing scRNA sequencing of the 3 vehicle controls alongside the 3 PD1 -inhibitor treated mice, we performed standard quality control and data preprocessing steps to arise at a cleaned and annotated dataset. Then through multiple rounds of cell type annotation we were able to identify and subcluster the LASP population into two distinct groups: LASP Canonical (marked by Aldh1 a3, Kit, Cd14, Fcgbp and Lurapl l) and LASP Aberrant (marked by Csn2, Csn1s1 , Elf5, Lalba, Cdknl a, Cdkn2a, Trp63, Krt5, Col3a1 , Col1 a1). We found that the PD1 inhibitor treated samples had reduced ratio of LASP Aberrant to LASP Canonical cells in comparison to the vehicle control Brcal mice (Figure 8a). This is further supported by an overall reduction in Csn2 expression in LASPs from the PD1 treatment group compared to vehicle control. These results strongly support the efficacy of PD1 treatment in enhancing the targeting of potentially malignant aberrant LASP cells in a pre-cancer setting. Further survival studies in these mouse lines will reveal the eventual effect of these changes in cellular dynamics on tumour formation rates.

Claims

CLAIMS1 . An immune checkpoint inhibitor for use in a method for the preventative treatment of a homologous recombination deficiency (HRD) associated cancer in a subject wherein the method comprises prophylactically administering one or more immune checkpoint inhibitor to said subject.

2. The immune checkpoint inhibitor for use according to claim 1 , wherein the preventative treatment is performed in a subject at risk of developing a HRD associated cancer.

3. The immune checkpoint inhibitor for use according to claim 1 or 2, wherein the subject comprises one or more alteration or aberration in one or more homologous recombination associated gene.

4. The immune checkpoint inhibitor for use according to any preceding claim, wherein the HRD associated cancer is a BRCA associated cancer, optionally wherein the subject comprises a BRCA pathogenic variant.

5. The immune checkpoint inhibitor for use according to claim 4, wherein the BRCA pathogenic variant comprises one or more mutation, alteration or aberration in BRCA1 and / or BRCA2.

6. The immune checkpoint inhibitor for use according to any preceding claim wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, a T cell immunoglobulin and ITIM domain (TIGIT) inhibitor, a V- domain Ig suppressor of T cell activation (VISTA) inhibitor, a BTLA inhibitor, a T-cell immunoglobulin and mucin domain-3 (TIM-3) inhibitor, a PD-L2 inhibitor, B7-H3 inhibitor, B7-H4 inhibitor, CD160 inhibitor, CD244 inhibitor, KLRG1 inhibitor, B7-1 inhibitor, B7-2 inhibitor, a KIR inhibitor, Galectin-9 inhibitor, NECTIN2 inhibitor, NECTIN3 inhibitor, an adenosine pathway inhibitor, an IDO inhibitor or a combination thereof.

7. The immune checkpoint inhibitor for use according to any preceding claim wherein the immune checkpoint inhibitor is selected from a molecule that targets PD-1 , PD-L1 and / or PD- L2.

8. The immune checkpoint inhibitor for use according to claim 7, wherein the molecule that targets PD-1 , PD-L1 and / or PD-L2 is used in combination with one or more further checkpoint inhibitor.

9. The immune checkpoint inhibitor for use according to any preceding claim, wherein the checkpoint inhibitor is selected from pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, dostarlimab, retifanlimab, ipilimumab, tremelimumab, relatlimab, trigolumab, domvanalimab.

10. The immune checkpoint inhibitor for use according to any preceding claim, wherein the HRD associated cancer is selected from breast cancer, ovarian cancer, prostate cancer, fallopian tube cancer, primary peritoneal cancer, stomach cancer, colorectal cancer, gall bladder cancer, cervical cancer, pancreatic cancer, lung cancer, head and neck cancer or melanoma.11 . The immune checkpoint inhibitor for use according to any preceding claim, wherein the HRD associated cancer is characterised by the presence of luminal adaptive secretory precursor cells (LASP).

12. The immune checkpoint inhibitor for use according to claim 11 , wherein the LASP comprise aberrant LASP.

13. The immune checkpoint inhibitor for use according to claims 1 1 or 12, wherein the LASP comprise increased expression of Csn2.

14. The immune checkpoint inhibitor for use according to any preceding claim, wherein the method further comprises: screening a biological sample obtained from said subject for the presence of aberrant LASP and / or increased Csn2 expression on one or more LASP.

15. The immune checkpoint inhibitor for use according to claim 14, wherein the presence of aberrant LASP and / or expression of Csn2 is assessed by RNA sequencing.

16. The immune checkpoint inhibitor for use according to any preceding claim, wherein the subject comprises an immune exhausted phenotype.

17. The immune checkpoint inhibitor for use according to any preceding claim, wherein the method further comprises: screening a biological sample from said subject for one or more cell comprising an immune exhausted phenotype.

18. The immune checkpoint inhibitor for use according to claim 14 or 17, wherein the biological sample is selected from blood, plasma, serum, sputum, bile, urine, tears and / or tissue.

19. The immune checkpoint inhibitor for use according to claim 14 or 17, wherein the biological sample is obtained from the breast, optionally a breast tissue sample.

20. The immune checkpoint inhibitor for use according to any one of claims 17 to 19, wherein the immune exhausted phenotype is characterised by an increased expression of PD-L1 , PD1 , CTLA4, LAG3, TIGIT, TIM3 VISTA, BTLA, TIM-3, PD-L2, B7-H3, B7-H4, CD160, CD244, KLRG1 , B7-1 , B7-2, KIR, Galectin-9, NECTIN2, NECTIN3, IDO or a combination thereof.21 . The immune checkpoint inhibitor for use according to any one of claims 17 to 20, wherein the one or more cell comprising an immune exhausted phenotype is selected from a CD8 T cell and / or a CD4 T cell.

22. The immune checkpoint inhibitor for use according to any one of claims 17 to 21 , wherein the one or more cell comprising an immune exhausted phenotype is selected from a CD8 TEM cell, a CD8 Tci cell, an NKT cell and / or an NK cell.

23. The immune checkpoint inhibitor for use according to any one of claims 17 to 22, wherein the immune exhausted phenotype is further characterised by an increased level of CD8 T cells, optionally CD8 Tci cells.

24. The immune checkpoint inhibitor for use according to any one of claims 17 to 23, wherein the one or more cell comprising an immune exhausted phenotype are localised to non-epithelial areas.

25. The immune checkpoint inhibitor for use according to any one of claims 17 to 24, wherein the one or more cell comprising an immune exhausted phenotype localised to non-epithelial areas, is characterised PD1 , CTLA4, LAG3, TIGIT, TIM3 VISTA, BTLA, TIM-3, PD-L2, B7-H3, B7-H4, CD160, CD244, KLRG1 , B7-1 , B7-2, KIR, Galectin-9, NECTIN2, NECTIN3, and / or IDO expression.

26. The immune checkpoint inhibitor for use according to any one of claims 17 to 25, wherein the one or more cell comprising an immune exhausted phenotype localised to non-epithelial areas, is characterised PD1 + / CD3+ expression.

27. The immune checkpoint inhibitor for use according to any preceding claim, wherein the immune checkpoint inhibitor is administered at a dose sufficient to restore cells to a non-immune exhausted phenotype.

28. The immune checkpoint inhibitor for use according to any preceding claim, wherein the immune checkpoint inhibitor is administered at a dose in the range of 0.1 ug / kg to 10 mg / kg.

29. The immune checkpoint inhibitor for use according to any preceding claim, wherein the wherein the checkpoint inhibitor is administered by intravenous infusion, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravesical, intradermal, topical or subcutaneous administration.

30. The immune checkpoint inhibitor for use according to any preceding claim, wherein one or more further preventative treatment of a HRD associated cancer is administered.

31. The immune checkpoint inhibitor for use according to claim 30, wherein the further preventative treatment of a HRD associated cancer is selected from surgery or small molecule therapy.

32. The immune checkpoint inhibitor for use according to claim 31 , wherein surgery comprises a mastectomy.

33. The immune checkpoint inhibitor for use according to claim 31 , wherein the small molecule therapy comprises tamoxifen, anastrozole, and / or raloxifene.

34. A method for the preventative treatment of a HRD associated cancer in a subject, wherein the method comprises prophylactically administering an immune checkpoint inhibitor to said subject.

35. A method of identifying a subject at risk of developing a HRD associated cancer comprising screening a biological sample, obtained from a subject, for one or more cell comprising an immune exhausted phenotype.

36. The method of claim 35, wherein the biological sample is selected from blood, plasma, serum, sputum, bile, urine, tears and / or tissue.

37. The method of claim 35 or claim 36, wherein the biological sample is a tissue sample, optionally a breast tissue sample.

38. The method of any one of claims 35 to 37, wherein the immune exhausted phenotype is characterised by an increased expression of one or more of the following markers: PD-L1 , PD1 ,CTLA4, LAG3, TIGIT, TIM3 VISTA, BTLA, TIM-3, PD-L2, B7-H3, B7-H4, CD160, CD244, KLRG1 , B7-1 , B7-2, KIR, Galectin-9, NECTIN2, NECTIN3, and / or IDO expression.

39. The method of any one of claims 35 to 38, wherein the one or more cell comprising an immune exhausted phenotype is selected from a CD8 T cell and / or a CD4 T cell.

40. The method of any one of claims 35 to 39, wherein the one or more cell comprising an immune exhausted phenotype is selected from a CD8 TEM cell, a CD8 Tci cell, an NKT cell and / or an NK cell.

41. The method of any one of claims 35 to 40, wherein the biological sample is further screened for an increased level of CD8 T cells, optionally Tci cells.

42. The method of any one of claims 35 to 41 , further comprising screening said biological sample for one or more cells comprising an immune exhausted phenotype localised in non- epithelial areas within the biological sample.

43. The method of any one of claims 35 to 42, further comprising screening said biological sample for PD1+ / CD3+ double positive cells localised in non-epithelial areas.

44. The method of any one of claims 35 to 43, wherein the method is performed in vitro or ex vivo.

45. The method of any one of claims 35 to 44, wherein the HRD associated cancer is a BRCA associated cancer.

46. The method of any one of claims 35 to 45, wherein the HRD associated cancer is selected from breast cancer, ovarian cancer, prostate cancer, fallopian tube cancer, primary peritoneal cancer, stomach cancer, colorectal cancer, gall bladder cancer, cervical cancer, pancreatic cancer, lung cancer, head and neck cancer or melanoma.

47. The method of any one of claims 35 to 46, wherein the subject is at risk of developing a HRD associated cancer48. The method of any one of claims 35 to 47, wherein the subject comprises one or more alteration or aberration in one or more homologous recombination associated gene.

49. The method of any one of claims 35 to 48, wherein the HRD associated cancer is a BRCA associated cancer, optionally wherein the subject comprises a BRCA pathogenic variant.

50. A method of identifying a subject at risk of developing a HRD associated cancer comprising: screening a biological sample, obtained from a subject, for one or more luminal adaptive secretory precursor cell (LASP) comprising an aberrant phenotype and / or increased expression of Csn2.

51. The method of claim 50, wherein the LASP comprise increased expression of Csn2.

52. The method of claim 50 or 51 , wherein the biological sample is screened for increased levels of aberrant LASP.

53. The method of claim 51 or 52, wherein the level of aberrant LASP and / or expression of Csn2 is assessed by RNA sequencing.

54. A method of identifying a subject as suitable for prophylactic treatment with one or more immune checkpoint inhibitor comprising selecting a subject, screening a biological sample obtained from said subject for the presence of an immune exhausted phenotype, selecting said subject for prophylactic treatment with one or more immune checkpoint inhibitor based on the presence of the immune exhausted phenotype.

55. A method of identifying a subject as suitable for prophylactic treatment with one or more immune checkpoint inhibitor comprising: selecting a subject, screening a biological sample obtained from said subject for the presence of one or more LASP comprising an aberrant phenotype and / or one or more LASP comprising increased expression of Csn2, and selecting said subject for prophylactic treatment with one or more immune checkpoint inhibitor.

56. A method of therapy monitoring, in a subject at risk of developing a HRD associated cancer, wherein the therapy comprises prophylactic treatment with one or more immune checkpoint inhibitor comprising: selecting a subject who has received or is receiving prophylactic treatment with one or more immune checkpoint inhibitor, and screening a biological sample obtained from said subject for the presence of one or more LASP comprising an aberrant phenotype and / or the expression level of Csn2 present on one or more LASP,wherein response to therapy is indicated by a reduction in the level of LASP comprising an aberrant phenotype and / or a decrease in expression of Csn2 on one or more LASP.

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