Methods for detecting breast cancer

Abstract

The present invention relates to a method for assessing the likelihood that a subject has breast cancer or a precancerous condition related to breast cancer. The present invention also relates to a method for determining whether treatment for breast cancer in a subject is necessary, and to a chemotherapeutic agent for use in treating a subject in need of such treatment.

Description

[Technical field]

[0001] The present invention relates to a method for assessing the likelihood that a subject has breast cancer or a precancerous condition associated with breast cancer. The present invention also relates to a method for determining whether treatment for breast cancer is required in a subject, and to a chemotherapeutic agent for use in treating a subject in need of such treatment. [Background technology]

[0002] Breast cancer is the most common malignant tumour diagnosed in women. It is reported that around 55,000 women are diagnosed with breast cancer each year in the UK and around 11,000 women die from the disease each year in the UK.

[0003] Breast cancer is a heterogeneous disease composed of several subtypes. Classification of breast cancer typically involves using immunohistochemical methods to determine the expression levels of several important hormone receptors associated with the development of breast cancer. Among the hormone receptors analyzed, the three most notable receptors are the progesterone receptor (PgR), the estrogen receptor (ER), and the human epidermal growth factor receptor 2 (HER2). Breast cancer patients who express PgR and ER and also have overexpression levels of HER2 typically exhibit better clinical outcomes due to the fact that they are more likely to respond well to hormone therapy, such as administration of chemotherapeutic agents that specifically target one or more of these important hormone receptors (e.g., Tamoxifen, Arimidex™ (anastrozole), Aromasin™ (exemestane), Femara™ (letrozole), Faslodex™ (fulvestrant), Herceptin™ (trastuzumab), Tykerb™ lapatinib).

[0004] One of the most aggressive subtypes of breast cancer, accounting for 15%-20% of all breast cancers, is triple-negative breast cancer (TNBC). TNBC is a type of breast cancer classified by the absence of protein expression of ER, PgR, and overexpression of HER2 protein. In the absence of PgR, ER, and HER2, TNBC tumors also do not respond to standard hormonal therapy treatment regimens, and therefore TNBC is usually associated with poor prognosis, low survival rates, and high rates of tumor recurrence. However, if diagnosed early, TNBC can be successfully treated with surgery, radiation therapy, and some chemotherapeutic agents, and can be further managed or prevented using selective mastectomy and / or prophylactic chemotherapy.

[0005] The prognosis for breast cancer patients, especially TNBC patients, is greatly improved if breast cancer is detected early. Therefore, there remains a need for simple, cost-effective methods to detect breast cancer, especially for detecting early onset of breast cancer in women at risk for TNBC. Summary of the Invention

[0006] According to a first aspect of the present invention there is provided a method of assessing the likelihood that a subject has breast cancer or a pre-cancerous condition associated with breast cancer, the method comprising analysing the breast of a subject to determine whether abnormally differentiated luminal progenitor cells are present in the breast, wherein the presence of the abnormally differentiated cells in the breast indicates that the subject is likely to have breast cancer or a pre-cancerous condition associated with breast cancer.

[0007] Suitably, the abnormally differentiated luminal progenitor cells are cells which abnormally differentiate to form alveolar structures (eg, milk-forming structures in the breast of a subject).

[0008] According to a second aspect of the present invention there is provided a method of determining whether a subject has breast cancer or a pre-cancerous condition associated with breast cancer, the method comprising analysing the breast of a subject to determine whether abnormally differentiated luminal progenitor cells and / or alveolar structures are present in the breast, wherein the presence of the abnormally differentiated cells and / or alveolar structures in the breast is indicative of the subject having breast cancer or a pre-cancerous condition associated with breast cancer.

[0009] According to a third aspect of the present invention there is provided a method of treating breast cancer in a subject, the method comprising: a) assessing the likelihood that a subject has breast cancer or a precancerous condition associated with breast cancer by a method described herein; b) if the subject is assessed as likely to have breast cancer or a precancerous condition related to breast cancer, administering to the subject a standard breast cancer treatment regimen.

[0010] According to a fourth aspect of the present invention there is provided a method of monitoring the onset of breast cancer or a pre-cancerous condition associated with breast cancer, or the recurrence and / or growth of breast cancer in a subject, the method comprising assessing, at two or more time points separated by a predetermined time interval, the likelihood that the subject has breast cancer or a pre-cancerous condition associated with breast cancer using a method defined herein.

[0011] According to a fifth aspect of the present invention there is provided a chemotherapeutic agent for use in the treatment of a subject in need of such treatment, the treatment comprising: a) assessing the likelihood that a subject has breast cancer or a precancerous condition associated with breast cancer by a method described herein; b) administering a chemotherapeutic agent to the subject if the subject is assessed as likely to have breast cancer or a precancerous condition related to breast cancer.

[0012] According to a sixth aspect of the present invention there is provided a method of assessing the likelihood that a subject has breast cancer or a pre-cancerous condition associated with breast cancer, the method comprising analysing a sample of material obtained from the subject's breast to determine whether beta-casein (CSN2) is present, wherein the presence of beta-casein (CSN2) in the sample of material obtained from the subject's breast is indicative of an increased likelihood that the subject has breast cancer or a pre-cancerous condition associated with breast cancer.

[0013] Where the assessment of the likelihood that the subject has breast cancer is performed with a high level of certainty, the method of the invention is a method of determining whether or not a subject has breast cancer.

[0014] Thus, in a preferred embodiment, the method of assessing the likelihood that a subject has breast cancer is a method of determining whether a subject has breast cancer. [Brief description of the drawings]

[0015] [Figure 1] Figure 1A) shows the percentage of cells in the luminal (left) or basal (right) compartment of mammary glands harvested from Bcl11a-labeled mice (e.g., mice treated with tamoxifen (TAM)) at 8 weeks post-labeling (8w) and 14.5 days of gestation (14.5dG) as determined by FACS, data are presented as mean ± SD and unpaired t-tests were performed (* indicates P<0.05). B) shows overviews of mammary epithelial structures (top two panels) and representative Z-slices (bottom two panels) as well as confocal 3D images of tdTomatopоs cells of CUBIC-cleared whole glands immunostained with EpCAM or K5, as indicated (scale bar, 100 µm). [Diagram 2] Figure 2A) is a schematic diagram of the MPA / DMBA protocol used to induce TNBC tumors in mice following induction of Bcl11a labeling, and B) is a confocal 3D image of tdTomatopоs cells of CUBIC-cleared whole glands immunostained for EpCAM or smooth muscle actin (SMA) as shown and represented as representative Z-slices of mammary epithelial structures (scale bar, 100 µm). [Diagram 3] Figure 3 shows Kaplan-Meier survival plots showing tumor-free survival of Brca1f / f;p53+ / -;Blg-Cre (Bcl11a+ / + or f / +) (n=14) and Bcl11af / f;Brca1f / f;p53+ / -;Blg-Cre (n=6). A log-rank (Mantel-Cox) test was performed (p=0.00017). [Figure 4] Figure 4 shows t-SNE representing mammary epithelial cells from Brca1f / f;p53+ / -;Blg-Cre (left panel) and Bcl11af / f;Brca1f / f;p53+ / -;Blg-Cre (right panel) mice isolated by FACS and subjected to droplet-based scRNAseq. Cells are colored based on their inferred cell type. [Diagram 5] FIG. 5 shows representative mammary whole mounts from virgin control, BCL11Aovx;MMTV-Cre, BCL11Aovx;Blg-Cre, Brcalf / f;p53+ / -;Blg-Cre and Bcl11af / f;Brca1f / f;p53+ / -;Blg-Cre mice from various ages. [Figure 6] Figure 6A) shows a graph representing the average protein expression of CSN2 in BRCA1 mutation carriers and non-BRCA1 carriers (normals) as measured by IHC, and B) shows a graph representing the average RNA expression of CSN2 in BRCA1 mutation carriers and non-BRCA1 carriers (normals) as measured by qPCR. [Figure 7]Figure 7A) shows a schematic diagram of 7 weeks after induction of Bcl11a labeling by a single intraperitoneal injection of low dose tamoxifen at maturity, mice were subjected to the MPA / DMBA protocol to induce TNBC tumors and samples were harvested 11 weeks later. B) shows tile scans of mammary gland sections immunostained with RFP (to detect tdTomatopos cells) and DAPI (scale bar, 100 μm). C) shows confocal 3D imaging of tdTomatopos cells in CUBIC-cleared whole glands immunostained with EpCAM (scale bar, 100 μm), shown as frame shots of 3D reconstructions of two representative mammary epithelial structures. D) shows mammary gland sections immunostained with RFP (to detect tdTomatopos cells), Csn2 or DAPI (scale bar, 100 μm). E) Schematic showing RNA extraction from FACS-isolated luminal progenitor cells at various time points after MPA / DMBA treatment, with a graph showing Csn2 expression in the various samples. [Figure 8] Figure 8 shows an extract from LC-MS analysis of mammary tissue from Brca1 / p53 mice. Figure 8 shows the mass spectrum of a peptide matching CSN2. [Figure 9] Figure 9 shows an extract from LC-MS analysis of mammary tissue from Brca1 / p53 mice. Figure 9 shows the mass spectrum of a peptide consistent with WAP. [Figure 10] Figure 10 shows an extract from an LC-MS analysis of blood from a lactating WT mouse. Figure 10 shows the mass spectrum of a peptide consistent with CSN2. [Figure 11] Figure 11 shows an extract from an LC-MS analysis of blood from a lactating WT mouse. Figure 11 shows the mass spectrum of a peptide consistent with WAP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Methods of the Invention The likelihood that a subject has breast cancer After extensive research, the present inventors have surprisingly discovered that luminal progenitor cells are abnormally differentiated in the breasts of both animal and human subjects diagnosed with breast cancer (e.g., TNBC), often before any visible signs of tumor development. Luminal progenitor cells are cells present in the female breast that normally only differentiate to form alveolar structures (e.g., milk-producing cells) during pregnancy. It was therefore surprising to the present inventors that such cells were found to be abnormally differentiated at an early stage of breast cancer (in non-pregnant subjects).

[0017] In light of this discovery, the present invention provides novel methods for determining the likelihood that a patient has breast cancer, using detection of abnormally differentiated luminal progenitor cells (and / or detection of alveolar structures) in the breast of a subject. Moreover, because differentiation of luminal progenitor cells, and the subsequent formation of alveolar structures, is typically observed before any visible signs of tumor formation, the present invention also advantageously provides methods for detecting early forms of breast cancer.

[0018] Thus, in one aspect of the invention, there is provided a method of assessing the likelihood that a subject has breast cancer or a precancerous condition associated with breast cancer, the method comprising analyzing the subject's breast to determine whether abnormally differentiated luminal progenitor cells are present, the presence of the abnormally differentiated cells being indicative of a high likelihood that the subject has breast cancer or a precancerous condition associated with breast cancer, and the absence of the abnormally differentiated cells being indicative of a low likelihood that the subject has breast cancer or a precancerous condition associated with breast cancer.

[0019] In certain embodiments, the methods include analyzing the subject's breast to determine whether abnormally differentiated luminal progenitor cells and / or alveolar structures are present in the breast, where the presence of the abnormally differentiated cells and / or the presence of alveolar structures indicates that the subject is likely to have breast cancer or a precancerous condition associated with breast cancer.

[0020] Suitably, the luminal progenitor cells are, for example, alveolar luminal progenitor cells which abnormally differentiate to form alveolar structures.

[0021] In certain embodiments, the methods of the invention include assessing the likelihood that a subject has breast cancer. Most suitably, the methods of the invention include assessing the likelihood that a subject has triple-negative breast cancer (TNBC).

[0022] In some embodiments, the methods of the invention include assessing the likelihood that a subject has a precancerous condition associated with breast cancer, a condition in which the morphology of certain cells is disturbed, thereby increasing the subject's risk of breast cancer.

[0023] breast analysis Analysis of the subject's breast to determine whether abnormally differentiated luminal progenitor cells and / or alveolar structures are present may include any suitable technique known in the art. In certain embodiments, analysis of the subject's breast includes scanning and / or imaging the breast to determine whether abnormally differentiated cells and / or alveolar structures are present. In other embodiments, the methods of the invention may include obtaining a sample of material from the subject's breast and analyzing the material to determine whether one or more species associated with abnormally differentiated cells are present.

[0024] Thus, in certain embodiments, analysis of the subject's breast comprises: a) scanning and / or imaging the breast to determine whether abnormally differentiated luminal progenitor cells are present; b) and / or obtaining a sample of material from the subject's breast and analyzing the material to determine whether species associated with abnormally differentiated luminal progenitor cells are present.

[0025] Scanning and / or imaging of the breast may be performed using any suitable technique known in the art. Suitably, scanning and / or imaging of the breast is performed using one or more techniques selected from magnetic resonance imaging (MRI), mammography, computed tomography (CT) scanning, and / or contrast imaging.

[0026] The species associated with the abnormally differentiated luminal progenitor cells may include, for example, proteins, DNA molecules and / or RNA molecules. Suitably, the species associated with the abnormally differentiated luminal progenitor cells is selected from the group consisting of milk proteins and / or DNA or RNA molecules encoding milk proteins.

[0027] In certain embodiments, the species associated with abnormally differentiated luminal progenitor cells is a milk protein. Suitably, the milk protein is selected from casein (e.g., beta-casein), alpha-lactalbumin, lactoferrin, immunoglobulin A, lysozyme, whey acidic protein (WAP) and serum albumin. Most suitably, the milk protein is casein (e.g., beta-casein) or whey acidic protein. Even more suitably, the milk protein is beta-casein or WAP. Most suitably, the milk protein is beta-casein.

[0028] In another embodiment, the species associated with abnormally differentiated luminal progenitor cells is a DNA or RNA molecule encoding a milk protein. Suitably, the DNA or RNA molecule encoding a milk protein is selected from alpha S1-casein (CSN1S1), beta-casein (CSN2), kappa-casein (CSN3), whey acidic protein and beta-lactoglobulin (LGB). More suitably, the DNA or RNA molecule encoding a milk protein is whey acidic protein (WAP) or beta-casein (CSN2). Most suitably, the DNA or RNA molecule encoding a milk protein is beta-casein (CSN2).

[0029] Analysis of material obtained from a subject's breast to determine whether species associated with abnormally differentiated luminal progenitor cells are present can be carried out using any suitable technique known in the art.

[0030] In embodiments in which analysis of material obtained from the subject's breast includes determining whether a protein is present, the analysis may be performed using one or more techniques selected from enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and mass spectrometry.

[0031] In embodiments in which analysis of material obtained from the subject's breast includes determining whether DNA or RNA molecules are present, the analysis may be performed using one or more techniques selected from quantitative polymerase chain reaction (qPCR), RNA sequencing (RNAseq), and DNA sequencing (DNAseq).

[0032] The sample of material from the subject's breast may be a sample obtained from the breast by biopsy or surgery, or a sample of material expressed from the breast (e.g., fluid expressed from the breast nipple), or a sample obtained from a sample of blood taken from the subject.

[0033] Suitably, the sample of material from the subject's breast is fluid expressed from the subject's breast. Most suitably, the sample of material from the subject's breast is fluid expressed via the nipple of the subject's breast. The sample of (expressed) material may be obtained using any technique known in the art, such as, for example, breast pump extraction and / or needle extraction.

[0034] Suitably, the sample of material from the subject's breast is a sample obtained from a blood sample taken from the subject. It will be understood that the sample of material from the subject's breast may be material that enters the subject's blood from the breast (e.g., from the mammary gland). Thus, the blood sample from which the sample of material from the breast is obtained does not necessarily have to be the subject's breast, but can be taken from anywhere in the subject's body.

[0035] In a particular embodiment, the method of the invention comprises analysing material obtained from the subject's breast to determine whether one or more additional species commonly associated with breast cancer and / or abnormally differentiated luminal progenitor cells are present or present at higher levels than in normal (control) subjects, as appropriate. The one or more additional species may, for example, be cancer genes commonly associated with breast cancer. Suitably, the one or more additional species are DNA or RNA molecules from genes selected from BCL11A, ELF5, SOX10, FOXC1 Ki-67, HOXD13 and PCDHGB7, and / or proteins expressed from said genes. Most suitably, the one or more additional species are selected from DNA or RNA molecules from genes selected from BCL11A, ELF5, SOX10, FOXC1 and Ki-67, and / or proteins expressed from said genes. Even more suitably, the one or more further species is selected from a DNA or RNA molecule from a gene selected from BCL11A, SOX10, and Ki-67, and / or a protein expressed from said gene. Most suitably, the one or more further species is a DNA or RNA molecule from BCL11A and / or Bcl11a protein.

[0036] The inventors have discovered that there is a strong correlation between the breast cancer oncogene BCL11A and aberrant differentiation of luminal progenitor cells. For example, deletion of Bcl11a confers protection against aberrant differentiation of luminal progenitor cells in the Brca1 / p53 TNBC mouse model (as described in the Examples below) and, in some cases, protection against breast tumor formation.

[0037] The inventors further discovered that many genes were upregulated in abnormally differentiated luminal progenitor cells, including Csn2, Csn1s1, B2m, Csn1s2a, H2.Q7, Bst2, Tceal9, Plin2, Muc15, Fth1, H2.K1, Mt1, Hspe1, Tmem176b, Mt2, Wfdc18, Sec61b, Vamp8, Tubb5, Ubd, Ogfrl1, Stmn1, Rbp1, H2.Q6, Dbi, Elf5, Psmb8, Chchd2, Serf2, Prdx2, Cair, Atp5h, Mgst1, Fam3c, H2afz, Pdk4, Ndufb9, Ubald2, Cox8a, Marcksl1, Cox6a1, H2.D1, Sod2, H3f3a, Tmem176a, Cox7b, Atp5o, Cox6b1, Psmb3, Pdia6, C3, Cox4i1, Ctsz, Gapdh, Mif, Atp5j2, P sma7, Clta, Sat1, Plscr1, Hspa8, Fkbp11, Scd1, Acot1, Mrps21, Cenpx, Cox5b, Cox6c, Pebp1, Ndufc2, Atp5c1, Ifitm3, Hspd1, Mbd1, DNA ja1, Pomp, Angptl4, Tecr, Ssr2, Cib1, C1qtnf1, Cldn3, Cd74, Cyba, P4hb, Map1lc3b, 1700025G04Rik, Calm2, Psmb1, Hsp90ab1, Eef1g, N dufb6, Atf3, Jpt1, Ndufa4, H2.Q4, Hsp90b1, Pdia3, Cuta, Fkbp2, Ssr4, H2afv, Ndufb11, Tmem258, Selenof, Cox5a, Sox4, Zfand5, mt.Co The genes we found, including 2, Prdx1, Atpif1, Uqcr10, Serp1, Nop10, Psmb4, Ndufa7, Ndufs6, Atp5j, Ndufa2, Rspo1, Taldo1, Ndufa11, Hspa5, Eci2, Uqcr11, Ndufb3, Eno1, Ehf, H2.T22, Cox7a2, Cks1b, Hist1h2bc, Npc2, Plscr2, Atp5g3, and Sdf2l1, were significantly upregulated in abnormally differentiated luminal progenitor cells.

[0038] Thus, in certain embodiments, the methods of the invention involve analyzing material obtained from the breast of a subject for the detection of the following genes: Csn2, Csn1s1, B2m, Csn1s2a, H2.Q7, Bst2, Tceal9, Plin2, Muc15, Fth1, H2.K1, Mt1, Hspe1, Tmem176b, Mt2, Wfdc18, Sec61b, Vamp8, Tubb5, Ubd, Ogfrl1, Stmn1, Rbp1, H2.Q6, Dbi, Elf5, Psmb8, Chchd2, Serf2, Prdx2, Cair, Atp5 h, Mgst1, Fam3c, H2afz, Pdk4, Ndufb9, Ubald2, Cox8a, Marcksl1, Cox6a1, H2.D1, Sod2, H3f3a, Tmem176a, Cox7b, Atp5o, Cox6b1, Psmb3, Pdia6 , C3, Cox4i1, Ctsz, Gapdh, Mif, Atp5j2, Psma7, Clta, Sat1, Plscr1, Hspa8, Fkbp11, Scd1, Acot1, Mrps21, Cenpx, Cox5b, Cox6c, Pebp1, Ndufc2, Atp5c1, Ifitm3, Hspd1, Mbd1, Dnaja1, Pomp, Angptl4, Tecr, Ssr2, Cib1, C1qtnf1, Cldn3, Cd74, Cyba, P4hb, Map1lc3b, 1700025G04Rik, Calm2 , Psmb1, Hsp90ab1, Eef1g, Ndufb6, Atf3, Jpt1, Ndufa4, H2.Q4, Hsp90b1, Pdia3, Cuta, Fkbp2, Ssr4, H2afv, Ndufb11, Tmem258, Selenof, Cox5a, The method includes determining whether one or more of Sox4, Zfand5, mt.Co2, Prdx1, Atpif1, Uqcr10, Serp1, Nop10, Psmb4, Ndufa7, Ndufs6, Atp5j, Ndufa2, Rspo1, Taldo1, Ndufa11, Hspa5, Eci2, Uqcr11, Ndufb3, Eno1, Ehf, H2.T22, Cox7a2, Cks1b, Hist1h2bc, Npc2, Plscr2, Atp5g3, and Sdf2l1 are present or upregulated.

[0039] Suitably, the method of the invention comprises analysing material obtained from the breast of a subject to determine whether one or more of the following genes are present or upregulated: Csn2, Csn1s1, B2m, Csn1s2a, H2.Q7, Bst2, Tceal9, Plin2, Muc15, Fth1, H2.K1, Mt1, Hspe1, Tmem176b, Mt2, Wfdc18, Sec61b, Vamp8, Tubb5, Ubd, Ogfrl1, Stmn1, Rbp1, H2.Q6, Dbi, Elf5 and Psmb8.

[0040] More suitably, the method of the invention comprises analysing material obtained from the breast of a subject to determine whether one or more of the following genes are present or upregulated: Csn2, Csn1s1, B2m, Csn1s2a, H2.Q7, Bst2, Tceal9, Plin2, Muc15, Fth1, H2.K1, Mt1, Hspe1, Tmem176b, Mt2, Wfdc18, Sec61b, Vamp8 and Tubb5.

[0041] Even more suitably, the methods of the invention include analysing material obtained from the breast of the subject to determine whether one or more of the following genes are present or upregulated: Csn2, Csn1s1, B2m, Csn1s2a, H2.Q7, Bst2 and Tceal9.

[0042] Most suitably, the methods of the invention comprise analysing material obtained from the breast of a subject to determine whether one or more of the following genes are present or upregulated: Csn2, Csn1s1, B2m and Csn1s2a.

[0043] The invention may also include a step of analysing material obtained from the breast of a subject to determine whether one or more proteins expressed from the above genes are present (or overexpressed).

[0044] In one embodiment, a method is provided for assessing the likelihood that a subject has breast cancer or a precancerous condition associated with breast cancer, the method comprising analysing a sample of material from the subject's breast obtained from a sample of blood taken from the subject to determine whether beta-casein (CSN2) is present, wherein the presence of beta-casein (CSN2) in the sample of material obtained from the subject's breast indicates that the subject is likely to have breast cancer or a precancerous condition associated with breast cancer.

[0045] Detection Technology As outlined above, the present invention provides a novel method for determining the possibility that a subject has breast cancer or a precancerous condition related to breast cancer.Therefore, the present invention can be beneficially used together with the existing methods available to physicians to assess the possibility that a subject has breast cancer (e.g., TNBC).Therefore, the present invention advantageously provides additional techniques that can be used to help physicians assess the possibility that a subject has breast cancer or a precancerous condition related to breast cancer.

[0046] Techniques commonly used by physicians to determine the likelihood that a subject has breast cancer are well known to those of skill in the art and include, for example, evaluation of the subject's medical history and / or the subject's family medical history, examination of the patient, imaging of all or part of the breast, testing the subject's blood for breast cancer biomarkers, genetic testing of the subject's DNA and / or RNA, and evaluation of a biopsy taken from the subject. Such techniques are routinely used to determine the presence of risk factors commonly associated with breast cancer (e.g., a family history of breast cancer, genetic defects / mutations commonly associated with breast cancer (e.g., mutations to the BRCA1 gene)), and / or atypical growths / tumors in the subject's breast).

[0047] In some embodiments, the methods of the present invention may include using one or more of the aforementioned techniques in addition to the methods described herein above, such that the presence of one or more risk factors is detected using one or more of the aforementioned techniques in addition to the presence of abnormally differentiated cellular and / or cellular structures in the subject's breast, which leads to an indication of whether the subject is likely to have breast cancer (e.g., TNBC).

[0048] subject It will be understood that the subject may be a human or animal subject. Preferably, the subject is a human subject and the breast cancer or precancerous condition is human breast cancer or a precancerous condition associated with human breast cancer.

[0049] In certain preferred embodiments, the subject is a non-pregnant subject. Thus, suitably, the subject is one who has had a pregnancy test performed, and most suitably, the subject is one who has had a pregnancy test performed and had a negative result.

[0050] In some embodiments, the subject is a subject who is identified as being at risk of developing triple-negative breast cancer.Accordingly, the subject is a subject who has one or more risk factors commonly associated with breast cancer (e.g., TNBC).The risk factors may include, for example, a family history of breast cancer, breast pain or discomfort, atypical growth on or around the breast, swelling or enlargement of one or both breasts, dense breast tissue, mutation of BRCA1 and / or BRCA2 genes, and homologous recombination repair deficiency.Accordingly, the subject is a subject who has a mutation of BRCA1 and / or BRCA2 genes, and most suitably, a mutation of BRCA1 gene.

[0051] Breast Cancer Treatment According to a third aspect of the present invention there is provided a method of treating breast cancer in a subject, the method comprising: a) assessing the likelihood that a subject has breast cancer or a precancerous condition associated with breast cancer by a method described herein; b) if the subject is assessed as likely to have breast cancer or a precancerous condition related to breast cancer, administering to the subject a standard breast cancer treatment regimen.

[0052] The one or more standard tumor treatment regimens can be any suitable treatment regimen commonly used to treat breast cancer or precancerous conditions related to breast cancer.Suitably, the one or more standard tumor treatment regimens are selected from surgery, radiation therapy, chemotherapy, immunotherapy, and combinations thereof.Most suitably, the standard tumor treatment regimen is selected from surgery, chemotherapy or radiation therapy, and combinations thereof.

[0053] Surgery can be a surgical intervention by a physician to remove all or part of a tumor (and / or breast). Non-limiting examples of suitable surgical interventions include biopsy, incision, drainage, and removal of the breast. Surgery also includes preventative surgical interventions, such as, for example, elective mastectomy.

[0054] Radiotherapy can be any form of treatment that utilizes ionizing radiation. Non-limiting examples of suitable radiotherapy treatments include, for example, external beam radiation therapy (EBRT), stereotactic radiosurgery (STRS) and external beam radiation therapy. Suitably, the radiotherapy is external beam radiation therapy (EBRT).

[0055] Chemotherapy can be treatment by administration of one or more anti-tumor (anti-cancer) agents. Non-limiting examples of suitable anti-tumor agents include, for example, tamoxifen, Arimidex™ (anastrozole), Aromasin™ (exemestane), Femara™ (letrozole), Faslodex™ (fulvestrant), Herceptin™ (trastuzumab), Tykerb™ (lapatinib), anthracyclines (e.g., doxorubicin or epirubicin), taxanes (e.g., docotaxel or paclitaxel), and combinations thereof. Suitably, the anti-tumor agent is an anthracycline (e.g., doxorubicin or epirubicin), taxanes (e.g., docotaxel or paclitaxel), or combinations thereof.

[0056] Chemotherapy may also include preventative chemotherapy and administration of agents such as tamoxifen and raloxifene that are commonly used to prevent and / or manage the development of breast cancer (eg, TNBC).

[0057] Immunotherapy can be any form of treatment that utilizes the patient's immune system. Non-limiting examples of suitable immunotherapy treatments include, for example, treatments that utilize one or more of monoclonal antibodies (MABs), vaccinations, cytokines, and CAR-T cells.

[0058] Active Surveillance Some breast cancers and precancerous conditions associated with breast cancer grow very slowly, and therefore, in such situations, active surveillance of the subject may be required to monitor the subject's breasts over time to see if breast cancer (or precancerous conditions associated with breast cancer) forms and / or grows.

[0059] Active surveillance is particularly useful for subjects identified as being at risk for developing breast cancer (e.g., triple-negative breast cancer), such as, for example, subjects with mutations in the BRCA1 and / or BRCA2 genes or a family history of breast cancer, or subjects who are homologous recombination repair deficient.

[0060] Thus, according to a fourth aspect of the present invention there is provided a method of monitoring the onset of breast cancer or a pre-cancerous condition associated with breast cancer, or the recurrence and / or growth of breast cancer in a subject, the method comprising assessing, at two or more time points separated by a predetermined time interval, the likelihood that the subject has breast cancer or a pre-cancerous condition associated with breast cancer by a method defined herein.

[0061] It will be understood that the number and duration of the predetermined time intervals may vary depending on the intended use of the method.For example, the predetermined time intervals used may vary depending on the type of breast cancer to be detected, the type and sensitivity of the instrument used in the analysis, and / or the subject on which the analysis is being performed.Such variations in the predetermined time intervals can be routinely determined by those skilled in the art based on the intended use of the methodology.

[0062] In certain embodiments of the methods for monitoring breast cancer or a precancerous condition associated with breast cancer, or for monitoring the recurrence and / or growth of breast cancer in the present subject matter, the predetermined time interval is every 1-12 months (e.g., every 1-6 months, every 1-3 months, every 1-2 months, or every month).

[0063] In other embodiments of the methods for monitoring breast cancer or a precancerous condition associated with breast cancer, or for monitoring the recurrence and / or growth of breast cancer in the subject matter of the present invention, the predetermined time interval is every 1-4 weeks (e.g., every 1-3 weeks, every 1-2 weeks, or every week).

[0064] In other embodiments of the methods for monitoring breast cancer or a precancerous condition associated with breast cancer, or for monitoring the recurrence and / or growth of breast cancer in the subject matter of the present invention, the predetermined time interval is every 1-14 days (e.g., every 1-7 days, every 1-3 days, or every day).

[0065] Chemotherapeutic agents According to a fifth aspect of the present invention there is provided a chemotherapeutic agent for use in the treatment of a subject in need of such treatment, the treatment comprising: a) assessing the likelihood that a subject has breast cancer or a precancerous condition associated with breast cancer by a method described herein; b) if the subject is assessed as likely to have breast cancer or a precancerous condition related to breast cancer, administering a chemotherapeutic agent to the subject.

[0066] In certain embodiments, the chemotherapeutic agent is selected from tamoxifen, Arimidex™ (anastrozole), Aromasin™ (exemestane), Femara™ (letrozole), Faslodex™ (fulvestrant), Herceptin™ (trastuzumab), Tykerb™ (lapatinib), anthracyclines (e.g., doxorubicin or epirubicin) or taxanes (e.g., docotaxel or paclitaxel). Suitably, the chemotherapeutic agent is an anthracycline (e.g., doxorubicin or epirubicin) or a taxane (e.g., docotaxel or paclitaxel).

[0067] The preferred and suitable features related to the first aspect of the invention described herein above also represent preferred and suitable features of all further aspects of the invention. Thus, any feature, integer, property, or means described in connection with a particular aspect, embodiment, or example of the invention should be understood to be applicable to all aspects, embodiments, or examples described herein, unless inconsistent. All features disclosed in this specification (including the accompanying claims, abstract, and drawings), and / or all steps of any method so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0068] The preferred and suitable features related to the first aspect of the invention described herein above also represent preferred and suitable features of all further aspects of the invention. Thus, any feature, integer, property, or means described in connection with a particular aspect, embodiment, or example of the invention should be understood to be applicable to all aspects, embodiments, or examples described herein, unless inconsistent. All features disclosed in this specification (including the accompanying claims, abstract, and drawings), and / or all steps of any method so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Item 1 1. A method for assessing the likelihood that a subject has breast cancer or a precancerous condition associated with breast cancer, the method comprising analyzing the subject's breast to determine whether abnormally differentiated luminal progenitor cells are present in the breast, wherein the presence of the abnormally differentiated cells in the breast indicates that the subject is likely to have breast cancer or a precancerous condition associated with breast cancer. Section 2 Item 2. The method according to item 1, wherein the abnormally differentiated cells abnormally differentiate to form cystic structures. Section 3 The method of any one of paragraphs 1 to 2, wherein the method comprises analyzing the subject's breast to determine whether follicular structures are present in the subject's breast. Section 4 Item 4. The method according to any one of Items 1 to 3, wherein the subject is identified as being at risk of developing triple-negative breast cancer. Section 5 The method according to any one of items 1 to 4, wherein the subject has a mutation in the BRCA1 and / or BRCA2 gene, for example, a mutation in the BRCA1 gene, or has a family history of breast cancer, or is homologous recombination repair deficient. Section 6 analyzing the subject's breast, a) scanning and / or imaging the breast to determine whether abnormally differentiated cells are present; and / or b) obtaining a sample of material from the subject's breast and analyzing the material to determine whether species associated with abnormally differentiated cells are present. The method of any one of claims 1 to 5, comprising one or more techniques selected from the following: Section 7 7. The method of claim 6, wherein the species associated with the abnormally differentiated cell is selected from the group consisting of a protein, a DNA molecule, and an RNA molecule. Section 8 8. The method of claim 7, wherein the species is selected from the group consisting of milk proteins and DNA or RNA molecules encoding milk proteins. Section 9 9. The method of claim 8, wherein the milk protein is selected from casein (e.g., beta-casein), alpha-lactalbumin, lactoferrin, immunoglobulin immunoglobulin A, lysozyme, whey acidic protein and serum albumin. Section 10 10. The method of claim 9, wherein the milk protein is selected from beta-casein and whey acidic protein. Section 11 11. The method of claim 10, wherein the milk protein is beta-casein. Item 12 Item 11. The method according to item 10, wherein the milk protein is whey acidic protein. Item 13 Item 13. The method of any one of items 8 to 12, further comprising analyzing material obtained from the subject's breast to determine whether additional species associated with abnormally differentiated cells are present. Item 14 14. The method of claim 13, wherein the further species associated with the abnormally differentiated cell is DNA or RNA from a gene selected from BCL11A, ELF5, SOX10, FOXC1 ki-67, HOXD13 and PCDHGB7, and any protein expressed from these genes. Item 15 Item 15. The method according to any one of items 7 to 14, wherein the presence of protein in the sample is determined using one or more techniques selected from enzyme-linked immunosorbent assay (ELISA), Western blot analysis and mass spectrometry, and / or the presence of DNA or RNA in the sample is determined using one or more techniques selected from quantitative polymerase chain reaction (qPCR), RNA sequencing (RNAseq) and DNA sequencing (DNAseq). Section 16 Item 16. The method according to any one of items 6 to 15, wherein the sample of material from the subject's breast is obtained via the nipple of the subject's breast or from a surgical procedure or biopsy. Section 17 Item 17. The method according to any one of items 6 to 16, wherein the sample is selected from fluid expressed from the breast of the subject. Section 18 Item 18. The method of any one of items 6 to 17, wherein the sample of material is obtained from the subject's breast via a breast pump and / or needle. Section 19 Item 16. The method according to any one of items 6 to 15, wherein the sample of material from the breast is obtained from a sample of blood taken from the subject. Section 20 20. The method of claim 19, wherein the blood sample is not taken from the breast of the subject. Section 21 21. The method of claim 19 or 20, wherein the sample of material from the breast of the subject is obtained from a sample of blood taken from the subject, and the species associated with the abnormally differentiated cells is beta-casein (CSN2). Section 22 7. The method of claim 6, wherein the subject's breast is scanned and / or imaged using one or more techniques selected from magnetic resonance imaging (MRI), mammography, computed tomography (CT) scanning, and / or contrast imaging. Section 23 Item 23. The method according to any one of items 1 to 22, wherein the subject has undergone a pregnancy test. Section 24 1. A method of treating breast cancer, comprising: a) assessing the likelihood that the subject has breast cancer or a precancerous condition associated with breast cancer by a method as claimed in any one of claims 1 to 23; b) if the subject is assessed as likely to have breast cancer or a precancerous condition related to breast cancer, administering to the subject a standard breast cancer treatment regimen. Section 25 25. The method of claim 24, wherein the standard breast cancer treatment regimen is selected from surgery, chemotherapy, and / or radiation therapy. Section 26 25. The method of claim 24, wherein the standard breast cancer treatment regimen is selected from elective mastectomy and / or preventive chemotherapy. Section 27 A method for monitoring the onset of breast cancer or a precancerous condition associated with breast cancer in a subject, or for monitoring the recurrence and / or growth of breast cancer, said method comprising assessing, at two or more time points separated by a predetermined time interval, the likelihood that said subject has breast cancer or a precancerous condition associated with breast cancer by a method as claimed in any one of claims 1 to 23. Section 28 1. A chemotherapeutic agent for use in the treatment of a subject in need thereof, said treatment comprising: a) assessing the likelihood that the subject has breast cancer or a precancerous condition associated with breast cancer by a method as claimed in any one of claims 1 to 23; b) administering the chemotherapeutic agent to the subject if the subject is assessed as likely to have breast cancer or a precancerous condition related to breast cancer. Section 29 29. The chemotherapeutic agent according to item 28, wherein the chemotherapeutic agent is an anthracycline (e.g., doxorubicin or epirubicin) or a taxane (e.g., docotaxel or paclitaxel).

[0069] Working Example Materials and Methods Mouse experiments All experimental animal work was performed in accordance with the Animals (Scientific Procedures) Act 1986 (UK) and approved by the Sanger Institute Ethics Committee. f / f ;p53 + / - Blg-Cre (JAX012620) (J Pathol., 211, 389-398 (2007)) were used to study the formation of TNBC tumors. These mice express Bcl11a f / f Mating it with mice expressing Bcl11a f / f , Brca1 f / f ;p53 + / -We generated Blg-Cre mice. Bcl11a knockout in luminal progenitor cells resulted in the expression of Bcl11a f / f This was achieved by crossing the Rosa26-LSL-BCL11A gene with a Blg-Cre mouse (Transgenic Res., 7, 387-396 (1998)). ovx Mice have been recently described (Nat. Commun., 0, 3327, (2018)). Briefly, the ROSA26 allele is targeted by a construct containing the cDNA of human BCL11A, preceded by a loxP-flanked STOP cassette and marked eGFP, under the control of an internal ribosome entry site (IRES) downstream of the inserted cDNA, and transcription of the transgene is controlled by the CAGG promoter. These mice were then transformed with Brca1 f / f ;p53 + / - Blg-Cre to study the effect of BCL11A overexpression on TNBC tumorigenesis. CreERT2 The alleles were generated by introducing an IRES-CreERT2-polyA-FRT-PGK-E7-Neo-polyA-FRT cassette into the 3'UTR of the Bcl11a locus. Gene targeting was performed in AB2.2 cells (129 background). Correct targeting was confirmed by long-range PCR, and targeted clones were used for blastocyst injections. Chimeric mice were then mated with wild-type mice (C57 / B6) to confirm germline transmission. Bcl11a CreERT2 The strain was the Rosa26-LSL-tdTomato mouse strain (JAX007905). 25 The animals used in each experiment were Bcl11a CreERT2Mice were heterozygous for Rosa26-LSL-tdTomato and homozygous for Rosa26-LSL-tdTomato. Lineage tracing was induced at maturity (5 weeks of age) or adulthood (9 weeks of age) by one or three intraperitoneal injections of tamoxifen (1 mg per injection) in corn oil on three consecutive days. Tamoxifen administration was confirmed by qRT-PCR to result in bona fide labeling of Bcl11a-expressing cells in the mammary gland. Tissues were collected at the indicated time points after injection. For pregnancy, females were mated to studs 8 weeks after tamoxifen injection and tissues were collected at 14.5 days of gestation. At each time point, Bcl11a CreERT2 Wild-type control littermates were injected simultaneously with the experimental animals and tissues were harvested to establish background levels of tdTomato fluorescence in both FACS and whole-tissue IF experiments. The scRNA-seq experiment was set up to allow all animals to be harvested and tissues processed simultaneously, with two individual mice per genotype used for the study. The estrous cycle stage of nulliparous animals was determined by vaginal smears. All mice were housed in individually ventilated cages under a 12:12-h light-dark cycle, had free access to water and food, and were eventually euthanized by anesthesia.

[0070] Isolation of mammary glands into single cell suspensions Mammary glands (excluding the cervical pair) with lymph nodes removed were dissected from mice and resuspended in DMEM / F12 (Gibco) + 10 mM HEPES (Gibco) + 1 mg ml -1 Collagenase (Roche) + 100Uml -1 The cells were digested with hyaluronidase (Sigma) (CH) + gentamicin (Gibco) O / N at 37 °C. For scRNA-seq experiments, mammary glands were instead mechanically dissociated after harvesting and the minced tissue was transferred to digestion mix containing 2 volumes of CH for 3 h at 37 °C, vortexing every 30 min. After lysis of red blood cells with NH4Cl, the cells were digested with warm 0.05% trypsin-EDTA (Gibco), 5 mg ml -1 Dispase (Sigma) and 1 mg ml -1 The mixture was briefly digested with DNase (Sigma) and filtered through a cell strainer (BD Biosciences).

[0071] Cell labeling followed by flow cytometry and sorting Single cell suspensions were incubated in HF medium (Hank's balanced salt solution (Gibco) + 1% fetal bovine serum, Gibco) + 10% normal rat serum (Sigma) for 20 min on ice to preblock prior to antibody staining. All antibody incubations were performed for 10 min on ice in HF medium. Mouse mammary cells were stained with the following primary antibodies: Cd31-biotin (eBioscience, clone 390, 1 μg ml -1 , 1:500); Cd45-biotin (eBioscience, clone 30F11, 1 μg ml -1 , 1:500); Ter119-biotin (eBioscience, clone Ter119, 1 μg ml-1, 1:500), EpCAM-APC / Cy7 (Biolegend, clone G8.8, 0.5 μg ml -1 , 1:500), Cd49f-BV421 (Biolegend313623, 2 μg ml -1 , 1:100), Cd49b-AF488 (Biolegend, clone HMa2, 1 μg ml -1 , 1:500) and Sca1-AF647 (Biolegend, clone D7, 1 μg ml -1 The cells were then stained with streptavidin-APC or streptavidin-PE / Cy7 (BD-Biosciences, 0.4 μg ml -1 , 1:500). 7-AAD (Sigma, 10 μg ml -1 Human mammary gland cells were incubated with the following primary antibodies: CD45-APC (Biolegend, clone H130, 1:100), CD31-APC (Biolegend, clone WM-59, 1:100), EPCAM-APC / Fire750 (Biolegend, clone 9C4, 1:50), CD49f-PE / Cy7 (Biolegend, clone GoH3, 1 μg ml -1, 1:200). DAPI was used to detect dead cells. Before sorting, cells were filtered through a cell strainer (Partec). Cell sorting was performed using an SH800Z sorter (SONY) after sorting calibration was performed using the automated setup beads (SONY) or FACS Aria Fusion immediately before sorting. Manual compensation was performed using single stained control cells. Gates were set using unstained cells and control animals. After doublets, dead cells and contaminating hematopoietic, endothelial, and stromal cells were gated out. FACS data was analyzed using the SONY software on the SH800Z or FlowJo. Mouse cells were analyzed for tdTomato expression in the mammary luminal or basal compartment, or luminal cells were sorted for luminal progenitor CFC assays. For scRNAseq, EpCAM-positive cells were sorted in LoBind microcentrifuge tubes (Eppendorf) containing HF. After sorting, cells were spun down and resuspended in HF. Samples were counted manually using a modified Neubauer chamber and cell concentration was normalized by the addition of HF. Equal numbers of cells per sample were processed for scRNA library preparation. Samples were processed for library preparation within 9 hours of tissue isolation. Human luminal progenitor cells were sorted for RNA processing.

[0072] Luminal progenitor cell CFC assay For luminal progenitor cell colony formation assays, 5% fetal bovine serum (StemCell), 10 ng ml -1 Epidermal growth factor (Sigma), 10ng ml -1 Basic fibroblast growth factor (Peprotech), 4 μg ml -1 Luminal tdTomato was produced by irradiated feeders in EpiCult Medium (StemCell) supplemented with heparin, Pen Strep Glutamine (Gibco) and gentamicin (Sigma). pоs or tdTomato negCells were sorted and plated, and cultures were maintained at 37°C / 5% CO2 for 15 days, then fixed using ice-cold acetone / methanol (1:1) and visualized using Giemsa staining (Merck), and the number of mammary CFCs was enumerated.

[0073] MPA / DMBA Tumor Formation Protocol Seven weeks after tamoxifen administration, a single MPA sustained-release pellet (Innovative Research of America) was implanted subcutaneously into the mice. Mice were allowed to recover for 10 days and then orally administered 1 mg DMBA (Sigma). This was followed by three further doses of 1 mg DMBA weekly. Mice were then examined weekly for tumor incidence and harvested 11 weeks after the last DMBA dose. CreERT2 tdTom animals that did not receive tamoxifen but underwent the same tumor formation protocol, and littermates Bcl11a WT tdTom exposed to both tamoxifen and MPA / DMBA protocols showed no detectable tdTomato pos No cells were found to have any cytotoxic activity, confirming that labeling and proliferation of tdTomato cells was not an artifact of the MPA / DMBA protocol.

[0074] Optical tissue clearing and whole-mount immunostaining Mammary tissue was dissected and cut into large pieces (approximately 15 x 15 x 2 mm) for immunostaining and clearing. CUBIC-based tissue clearing was performed as previously described. 27(Breast Cancer Res.18,127(2016) and Nat. Commun.,7,13053(2016)) and combined with whole-mount immunolabeling for visualization of tdTomato cells. Briefly, CUBIC Reagent 1A was prepared using urea (Sigma, 10% (w / w)), N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine (Sigma, 5% (w / w)), triton-X100 (VWR, 10% (w / w)) and NaCl (Sigma, 25 mM) in distilled water. CUBIC Reagent 2 was prepared using sucrose (Fisher Scientific, 44% w / w), urea (Sigma, 22% w / w), 2,2',2''-nitrilotriethanol (Sigma, 9% w / w), and TritonX-100 (VWR, 0.1% w / w) in distilled water. Tissues were immersed in CUBIC Reagent 1A for 2–3 days at 37°C. For immunostaining, samples were washed and then blocked overnight at 4°C with PBS containing triton-X100 (0.5% (w / v)) and goat serum (10% (v / v)). Primary antibodies were diluted in blocking buffer for 4 days at 4°C with gentle rocking. Tissues were washed (3 × 1 h), incubated with Alexa Fluor-conjugated secondary antibodies for 2 days, washed with PBS, and transferred to CUBIC Reagent 2 for at least 1 day at 37°C to match the refractive index. Samples were immersed in CUBIC reagent 2 for imaging and imaged within 2 weeks. The following primary antibodies were used: rat anti-EpCAM-AF647 (Biolegend, clone G8.8, 1 μg ml -1 , 1:250), and rabbit anti-K5 (Covance, PRB160P, 1:100) were used for immunostaining. The following Alexa Fluor-conjugated secondary antibodies, goat anti-mouse 488 (A11001), and goat anti-rat 647 (A21247), were purchased from Thermo Fisher Scientific and used at 1:500.

[0075] Immunofluorescence Five-μm sections of mammary glands were stained with hematoxylin and eosin (H&E) or immunostained with antibodies against Csn2 (sc-166530, Santacruz, 1:50) and RFP (600-401-379, Rockland, 1:200). Secondary stains included goat anti-rabbit 647, goat anti-rat 647, or anti-mouse 488, anti-rabbit 594 (1:200, Invitrogen). Nuclear staining was detected using Prolong Gold Antifade Mountant with DAPI (Thermofisher, P36941).

[0076] Confocal microscopy and image analysis Images of whole-mount mammary glands were acquired using Leica TCS SP8 and SP5 inverted confocal microscopes equipped with 10× / 0.4 or 40× / 1.3 HC PL APO objectives. H&E images were acquired using a Zeiss Axioplan2 microscope. Laser power, line averaging, and step increments were manually adjusted to obtain optimal fluorescence intensity for each fluorophore while minimizing photobleaching. Image overviews and 3D reconstructions were generated using ImageJ software.

[0077] Differential expression Differential gene expression analysis was performed using edgeR (Bioinformatics, 26, 139-140 (2010)). For pairwise comparisons between clusters, genes with mean expression levels below 0.1 were excluded from the analysis. A negative binomial generalized log-linear model was fitted to the remaining genes with cluster assignment as covariate(s). The "treat" function with "trend=TRUE, robust=TRUE" was used to identify genes with a significantly higher log fold change than Iog2 (1.5) at an FDR of 0.01. Marker genes used for cell type inference were identified using the "findMarkers" function with default settings of scramble.

[0078] Western blot Cells were lysed using RIPA (Cell signalling) and protease inhibitors (Roche) according to the manufacturer's instructions. For tissue lysis, mammary tissue was ground using liquid nitrogen, a mortar and pestle. After the ground samples were lysed in RIPA, the samples were passed through a 25G needle. Total protein was measured using the bicinchoninic acid (BCA) method (Pierce Biotechnology). In total, 50 μg of cell lysate was resolved using a 7.5% SDS-PAGE gel and transferred to a PVDF membrane by electroblotting. The membrane was blocked with 5% (w / v) milk in Tris-buffered saline with 0.1% Tween-20 (TBST). Blots were then incubated with primary antibodies overnight at 4 °C, washed with TBST, and subsequently probed with secondary antibodies for 1 h at room temperature, as indicated. ECL solution was then added to the membrane for analysis. The antibodies used were BCL11A (A300-380A, Bethyl, 1:1000), WAP (sc-398276, Santa Cruz, 1:1000) and β-actin (sc-130656, Santa Cruz, 1:1000).

[0079] Liquid Chromatography Mass Spectrometry (LC-MS) An equal volume of 6M GuHCI was added to the serum and mixed. Organic solvent (75% ACN) was then added and the sample mixed. The samples were centrifuged, the supernatant decanted and evaporated under OFN. The extract was reconstituted in 0.1% formic acid, loaded onto a Waters HLB Micro elution plate and extracted by SPE. The eluent was evaporated, reduced and alkylated, then digested and analyzed by LC-MS using a nanoLC system. Peptides were analyzed using a Q-Exactive Plus orbitrap using PRM and IDA-based analysis.

[0080] statistical significance All P values ​​were calculated using Student's t test unless otherwise indicated in the figure legends.

[0081] result The role of Bcl11A in vacuole formation To assess whether Bcl11a-expressing cells are part of blast differentiation and TNBC tumor initiation, we generated Bcl11a reporter mice by knocking in an IRES-CreERT2 cassette into the 3'UTR region of Bcl11a, herein referred to as Bcl11a CreERT2 These mice, when crossed with Rosa26-LSL-tdTomato reporter mice (tdTom), allowed us to perform lineage tracing. Tamoxifen-mediated CreERT2 activation induced the expression of tdTomato in Bcl11a-expressing cells and any of their future progeny (Figure 1A). Fluorescence-activated cell sorting (FACS) analysis 1 day after labeling revealed that tdTomato pos The majority of cells (95% ± 1.6) were located within the luminal compartment and appeared to be primarily luminal progenitor cells (Figure 1A).

[0082] To investigate whether Bcl11a-labeled cells contribute to the alveolar formation process, females were mated 8 weeks after tamoxifen injection and mice were analyzed at 14.5 days of gestation. pos We found that the number of luminal cells increased significantly, constituting approximately 30% of the luminal compartment, and analysis after natural weaning revealed persistence of labeled cells at levels similar to those in nulliparous mice ( Fig. 1B ).

[0083] The above data support a role for Bcl11a in alveolar production.

[0084] Expression of Bcll11a is detectable in early stages of tumor development in TNBC models Long-lived tdTomato in the early stages of TNBC development pоs To investigate the cellular role of Bcl11a CreERT2tdTom mice were subjected to the MPA / DMBA protocol (Figure 2A) for TNBC tumor formation (Genome Biol., 8, R76 (2007) and Nature, 468, 103-107 (2010)), which has previously been shown to be Bcl11a dependent (Nat. Med., 15, 907-913 (2009)). Tile scans of glands at the pre-cancerous stage 11 weeks after DMBA administration show that tdTomato pos Cellular enlargement and the appearance of cyst-like structures were evident, and in some cases, this enlargement led to the formation of abnormal structures that grew and invaded the lumen (FIG. 2B).

[0085] Bcll11a knockout / out mice do not develop TNBC To investigate how driver oncogenes regulate the fate of luminal progenitor cells in TNBC, we developed a Brca1 / p53 TNBC mouse model that contains conditional Brca1 loss of function in the luminal progenitor compartment (Brca1 f / f ;p53 + / - ;Blg-Cre) was transduced with the TNBC oncogene BCL11A 1 We focused on and used Bcl11a deletion in luminal progenitor cells, which was found to completely protect Brca1 / p53 mice from mammary tumor formation (Figure 1A), suggesting that Bcl11a is essential for Brca1-mediated TNBC.

[0086] RNASeq of mammary tissue identifies cyst-like cells in tumor-bearing mice (Bcl11A K / O) but not in disease-free animals (Bcl11A WT). RNASeq of mammary tissue from tumor (Bcl11a positive) and non-tumor (Bcl11a deficient) animals at week 30 (week 33 is prior to physical evidence of tumors). Clustering analysis reveals four common cell populations between tumor and non-tumor animals: Lp; luminal progenitor cells, Hsp; hormone-sensing progenitor cells, Hsd; hormone-sensing differentiated and Bsl; basal. In tumor-bearing animals, a population of Lp-Av (alveolar cells) was present.

[0087] This suggests that early in tumor formation (before any physical signs) there is a population of follicular cells that are not present in disease-free animals.

[0088] Overexpression of Bcl11A specifically in the mammary gland promotes alveolar formation, and these structures can be observed even in non-pregnant, early-stage tumor-bearing animals. To identify the cellular events that underpin this protective effect of Bcl11a deletion in the Brca1 / p53 model, we performed Brca1 / p53 and Bcl11a deletion in nulliparous mice at 30–33 weeks of age before tumors were detected. f / f We performed scRNAseq on mammary epithelial cells from Brca1 / p53 mice. We found that Brca1 / p53 mammary epithelium was phenotypically similar to that of pregnant mice (Figure 5), consistent with previous reports of precocious alveolar formation in a distinct model of Brca1 / p53 (Science (80-.) 314, 1467LP-1470 (2006)).

[0089] Bcl11a f / f , scRNAseq and whole-mount analysis of Brca1 / p53 mice revealed that deletion of Bcl11a completely abolished the aberrant differentiation behavior of luminal progenitor cells ( Fig. 5 ).

[0090] The above results suggest that Bcl11a-dependent cyst formation occurs at an early stage of TNBC.

[0091] Detection of abnormal differentiation in MPA / DMBA-treated animals Next, the long-lived tdTomato was expressed in the early stages of TNBC development. pоs We investigated the role of Bcl11a in cells. CreERT2 tdTom mice were subjected to the MPA / DMBA protocol (Figure 7A) for TNBC tumor formation (Genome Biol., 8, R76 (2007) and Nature, 468, 103-107 (2010)), which has previously been shown to be dependent on Bcl11a (Nat Commun., 6, 5987 (2015)). Tile scans of glands at the pre-cancerous stage 11 weeks after DMBA administration show that tdTomatopos The results revealed cell enlargement and the appearance of alveolar-like structures (Figure 7B). In some cases, the enlargement led to the formation of abnormal structures that grew and invaded the lumen (Figure 7C). 3D imaging of these structures revealed similarities to atypical ductal hyperplasia and ductal carcinoma in situ (Figure 7C). IHC analysis of these mice revealed the appearance of milk-producing cells that were compatible with the appearance of alveolar-like structures and consistent with the observations made in the Brca1 / p53 model (Figure 7D). RNA was extracted from FACS-isolated luminal progenitor cells at various time points after MPA / DMBA treatment. Figure 7E shows the expression of Csn2 in the various samples. MPA alone mice received MPA 3 weeks prior to sample collection. Quantification was performed by first normalizing to Gapdh levels compared to MPA alone samples.

[0092] The above results show that expression of Csn2 was observed in luminal progenitor cells 3 weeks after treatment, whereas tumors were not observed until approximately 12-14 weeks after treatment. Thus, these results demonstrate that the present invention, and in particular the detection of abnormally differentiated luminal progenitor cells and / or Csn2 in a subject's breast, is a superior method for detecting early signs of breast cancer (e.g., TNBC).

[0093] Detection of CSN2 and / or WAP Surprisingly, we found that LC-MS analysis of mammary tissue from Brca1 / p53 mice detects peptides matching CSN2 and WAP (Figures 8 and 9), correlating with the mice exhibiting aberrant differentiation of luminal progenitor cells described above.

[0094] Furthermore, using LC-MS, the same peptides could be detected in the blood of lactating WT mice, indicating that these proteins may enter the blood (Figures 10 and 11). These milk protein peptides are not detected (using LC-MS) in the serum of male mice. Thus, these peptides could be used for the early detection of abnormal luminal differentiation in females carrying Brca1 pathogenic variants (PV) using more sensitive methods.

[0095] RNA encoding CSN2 was also detected in the blood of Brca1 / p53 mice using qPCR. The same RNA was not detectable (using qPCR) in the blood of male mice.

[0096] Human data RNA was extracted from FACS-isolated mammary luminal progenitor cells from either i) control patients or ii) BRCA1-bearing patients. CSN2 expression in these human samples was then determined. Quantification was performed by first normalizing to GAPDH levels relative to the control STG218.

[0097] Figure 6 shows the average protein expression of CSN2 in BRCA1 mutation carriers and non-BRCA1 carriers (normals) as measured by IHC (Figure 6A) and the average RNA expression of CSN2 in BRCA1 mutation carriers and non-BRCA1 carriers (normals) as measured by qPCR (Figure 6B).

Claims

1. 1. A method of assessing the likelihood that a subject has breast cancer or a precancerous condition associated with breast cancer, the method comprising determining whether abnormally differentiated luminal progenitor cells are present in the breast by analyzing a sample of material from the subject's breast to determine whether a species associated with abnormally differentiated cells is present in the sample, the presence of the species associated with abnormally differentiated cells being indicative of a high likelihood that the subject has breast cancer or a precancerous condition associated with breast cancer, the species associated with abnormally differentiated cells being one or more of beta-casein (CSN2), a DNA molecule encoding beta-casein (CSN2), an RNA molecule encoding beta-casein (CSN2), whey acidic protein (WAP), a DNA molecule encoding whey acidic protein (WAP), and an RNA molecule encoding whey acidic protein (WAP). method.

2. 2. The method of claim 1, wherein the step of analyzing a sample of material from the subject's breast comprises determining the presence of proteins in the sample using one or more techniques selected from enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and mass spectrometry, and / or determining the presence of DNA and / or RNA molecules in the sample using one or more techniques selected from quantitative polymerase chain reaction (qPCR), RNA sequencing (RNAseq), and DNA sequencing (DNAseq).

3. 3. The method of claim 1 or 2, wherein the sample of material from the subject's breast is obtained from the nipple of the subject's breast or from a surgical procedure or biopsy.

4. The method of any one of claims 1 to 3, wherein the sample of material from the subject's breast is a sample obtained via a breast pump and / or a needle.

5. 5. The method of any one of claims 1 to 4, wherein the sample of material from the subject's breast is obtained from a sample of blood taken from the subject or is fluid expressed from the breast of the subject.

6. A method according to any one of claims 1 to 5, wherein the method comprises analysing the subject's breast to determine whether alveolar structures are present in the subject's breast.

7. The method of claim 6 , wherein the method comprises scanning and / or imaging the subject's breast to determine whether abnormal tissue is present in the subject's breast.

8. 8. The method of claim 7, wherein the subject's breast is scanned and / or imaged using one or more techniques selected from magnetic resonance imaging (MRI), mammography, computed tomography (CT) scanning, and / or contrast imaging.

9. 9. The method of any one of claims 1 to 8, wherein the method further comprises analysing a sample of material obtained from the subject's breast to determine whether additional species associated with abnormally differentiated cells are present in the sample.

10. 10. The method of claim 9, wherein the additional species associated with the abnormally differentiated cell is DNA or RNA from BCL11A, or any protein expressed from BCL11A.

11. The method of any one of claims 1 to 10, wherein the subject is identified as being at risk for developing triple-negative breast cancer.

12. The method of any one of claims 1 to 11, wherein the subject has a mutation in the BRCA1 and / or BRCA2 gene, or has a family history of breast cancer, or is homologous recombination repair deficient.

13. The method of any one of claims 1 to 12, wherein the subject is a non-pregnant subject.

14. 14. A method for monitoring the onset of breast cancer or a precancerous condition associated with breast cancer, or for monitoring the recurrence and / or growth of breast cancer in a subject, said method comprising carrying out a method according to any one of claims 1 to 13 at two or more time points separated by a predetermined time interval.

15. A chemotherapeutic agent for the treatment of breast cancer or a precancerous condition associated with breast cancer, characterized in that the subject of said treatment has been determined to have a high likelihood of having breast cancer or a precancerous condition associated with breast cancer by the method of any one of claims 1 to 13, said chemotherapeutic agent being Tamoxifen, Arimidex® (anastrozole), Aromasin® (exemestane), Femara® (letrozole), Faslodex® (fulvestrant), Herceptin® (trastuzumab), Tykerb® lapatinib), anthracycline, or taxane.

16. 16. The chemotherapeutic agent of claim 15, wherein the chemotherapeutic agent comprises an anthracycline or a taxane, the anthracycline being doxorubicin or epirubicin, and the taxane being docotaxel or paclitaxel.

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