Patent Application
20240310378
The present invention relates to a method of predicting the prognosis of a breast cancer patient by measuring the expression level of p53 and/or GATA3 in a lymph node metastasis sample.
This application claims the benefit of European patent application EP21156709.4, filed 11 Feb. 2021, which is fully incorporated herein by reference.
Molecular classification followed by targeted treatment has significantly improved prognosis of patients with breast cancer, yet one-third of breast cancer patients eventually develop distant metastases and succumb to disease. Although mortality is largely caused by metastases, the histologic and genetic analysis is performed on the primary tumour in current clinical care. Little is known about how disseminating cell phenotypes relate to the diverse cell types and microenvironments of the primary tumour or how these phenotypes relate to disease outcome.
The spread of tumour cells to the sentinel LNs is an important prognostic factor in breast cancer diagnosis and influences treatment decisions. Clinical classification of LN status, (nodal status) does not provide information about the biology of LN metastases, in contrast to measures such as tumour grade or molecular subtype, which are used to stratify primary tumours and also guide clinical decisions. To target disseminated tumour cells in the lymphatic system, node-positive breast cancer patients typically receive adjuvant chemotherapy. However, tumour grade and molecular subtype markers (ER, PR, HER2, and Ki-67) are not necessarily consistent through tumour progression or between primary and metastatic sites (Linstrom, L. S. 2012, J. Clin. Oncol. 30:2601). Further, some patients with high nodal status survive long term, whereas some node-negative patients do not, indicating that the amount of tumour cells in the LNs does not fully capture patient prognosis. Single-cell studies have shown substantial intra- and inter-tumour heterogeneity in primary tumours, which are associated with tumour type and distinct patient outcomes, but which single-cell phenotypes disseminate is unknown.
Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to predict breast cancer patient outcome using biomarkers present in LN metastases. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.
A first aspect of the invention provides a method to predict breast cancer patient prognosis by determining the expression level of the biomarker GATA3, and/or p53 in a lymph node metastasis sample. In some embodiments higher GATA3, or lower p53 expression levels are associated with good prognosis, and lower GATA3, or higher p53 expression levels are associated with poor prognosis, and optionally, high and low biomarker expression level is based on useful expression level thresholds provided herein. In some embodiments the biomarker expression level is determined at the average expression level of a sample analysed in bulk, in other embodiments the biomarker expression level is measured at the level of a single cell, particularly using immunohistochemistry on a sample section.
A further aspect of the invention provides hormone-targeting antineoplastic drugs for use to treat patients assigned to good prognosis according to the first aspect of the invention, or aggressive, systemic, non-targeted antineoplastic drugs, or radiation for patients assigned to a poor prognosis. In another embodiment, the present invention relates a pharmaceutical composition comprising at least one of the antineoplastic compounds of the present invention, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent or excipient. A kit, or a system providing labelled molecular probes to measure GATA3 and/or p53 for analysis of a lymph node metastasis are further encompassed by the invention.
For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.
The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.
The term p53 in the context of the present specification relates to the tumour suppressor protein cellular tumour antigen p53 (Uniprot P04637), encoded by the gene TP53, also known as phosphoprotein p53.
The term GATA3 in the context of the present specification relates to Trans-acting T-cell specific transcription factor GATA-3 (Uniprot P23771), encoded by the GATA3 gene.
The term patient according to the invention refers to a subject who has been diagnosed with breast cancer tumour, in other words a malignant neoplasm originating in breast tissue, specifically where the tumour has spread to lymph node (LN) tissue.
The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes—and products thereof—of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.
The term expression level as used herein refers to a quantitative measure of gene expression, at either the mRNA or protein level. If the word “expression” is used herein in the context of “gene expression” or “expression of a marker or biomolecule” and no further qualification of “expression” is mentioned, this implies “positive expression”. In the present specification, the term high, or positive when used in the context of expression of a biomarker unless specified otherwise, refers to expression level of biomarker relative to a negative control. The negative control is used to identify a background signal threshold level the label's signal when bound to a structure (for example, a cell, or a tissue lysate, or mRNA preparation) for a biomarker, and cells, or samples providing a signal above this background level in the patient sample are classified as high or positive for the biomarker. Expression level may be measured either at the level of a bulk analysis of sample, or at the level of a single cell, and optional thresholds to identify positive or high expression in these formats are provided herein.
The term negative control as used herein refers to a sample processed alongside the patient sample, which does not express the biomarkers, for example, a LN or tumour sample previously determined to lack p53 and/or GATA3 expression, or a healthy tissue. Suitable negative controls according to the invention include molecular probe controls, wherein a portion of the patient sample is assayed lacking the molecular probes for p53 and/or GATA3, or where the molecular probe has been exchanged for an isotype control. In particular embodiments, the biomarker expression level in the sample is compared to a healthy tissue negative control sample, either from the patient, or more particularly, from a healthy subject. A healthy liver sample to be analysed in a tissue section, or microarray such as that used in the examples as can be obtained commercially (Novusbio).
The term molecular probe in the context of the present specification relates to a specific ligand sensitive to expression of an antigen, particularly p53 and GATA3, at the level of mRNA, or protein expression.
In embodiments relating to biomarker assessment at the protein level, molecular probes of particular use include an antibody, antibody fragment, an antibody-like molecule or aptamer, more particularly an antibody or antibody fragment, that can bind to a target molecule with a dissociation constant of ≤10−7 mol/l, particularly ≤10−8 mol/l. In other embodiments relating to biomarker mRNA expression level measurements, molecular probes of particular use are primers, complimentary nucleic acids probes specific for p53 or GATA3 mRNA, suitable for quantitative measurements of the amount of mRNA, or cDNA copies of the p53 or GATA3 gene present in sample the using polymerase chain reaction (PCR), or in situ hybridisation assays. The molecular probe according to the invention comprises a detectable marker such as a particle, bead, dye or enzyme, and must be paired with an assay sensitive to difference in the amount of molecular probe on a sample, and a control negative sample.
In the context of the present specification, the term antibody refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen binding fragment or single chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Similarly, the term encompasses a so-called nanobody or single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain, or a multimeric antibody fragment molecule, or single chain variable fragment, fusion proteins of two or more variable regions connected with linker peptides.
The term antibody-like molecule in the context of the present specification refers to a molecule capable of specific binding to another molecule or target with high affinity/a Kd≤10E-8 mol/l. An antibody-like molecule binds to its target similarly to the specific binding of an antibody. The term antibody-like molecule encompasses a repeat protein, such as a designed ankyrin repeat protein (Molecular Partners, Zürich), an engineered antibody mimetic proteins exhibiting highly specific and high-affinity target protein binding (see US2012142611, US2016250341, US2016075767 and US2015368302, all of which are incorporated herein by reference). The term antibody-like molecule further encompasses, but is not limited to, a polypeptide derived from armadillo repeat proteins, a polypeptide derived from leucine-rich repeat proteins and a polypeptide derived from tetratricopeptide repeat proteins.
The term antibody-like molecule further encompasses a specifically binding polypeptide derived from a protein A domain, a fibronectin domain FN3, a consensus fibronectin domain, a lipocalin (see Skerra, Biochim. Biophys. Acta 2000, 1482(1-2):337-50), a polypeptide derived from a Zinc finger protein (see Kwan et al. Structure 2003, 11(7):803-813), a Src homology domain 2 (SH2) or Src homology domain 3 (SH3), a PDZ domain, a gamma-crystallin, ubiquitin, a cysteine knot polypeptide or a knottin, cystatin, Sac7d, a triple helix coiled coil (also known as alphabodies), a Kunitz domain or a Kunitz-type protease inhibitor and a carbohydrate binding module 32-2.
The term specific binding in the context of the present invention refers to a property of ligands that bind to their target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of ≤10−7 mol/L when binding to its target, but a dissociation constant at least three orders of magnitude higher in its interaction with a molecule having a globally similar chemical composition as the target, but a different three-dimensional structure.
In the context of the present specification, the term labelled antibody or labelledmolecular probe is used for a molecular probe, particularly an antibody, or nucleic acid probe, being covalently bound to a detectable label. The expression of GATA3, or p53 may be assayed via techniques sensitive to the probe/detectable label chosen, such as PCR, fluorescence microscopy, flow cytometry, mass cytometry, immunohistochemistry or multiplex analyses.
Such detectable labels include those appropriate for flow cytometry detection systems, including, without limitation, small molecule capable of fluorescence in the visible or near infrared spectrum. Examples for fluorescent labels or labels presenting a visible color include, without being restricted to, fluorescein isothiocyanate (FITC), rhodamine, allophycocyanine (APC), peridinin chlorophyll (PerCP), phycoerithrin (PE), alexa Fluors (Life Technologies, Carlsbad, CA, USA), dylight fluors (Thermo Fisher Scientific, Waltham, MA, USA) ATTO Dyes (ATTO-TEC GmbH, Siegen, Germany), BODIPY Dyes (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene based dyes) and the like.
Such detectable labels include those appropriate for fluorescence immunohistochemistry, or in situ hybridisation detection methods, for example, without limitation, octadecyl rhodamine B, 7-nitro-2-1,3-benzoxadiazol-4-yl, 4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid, acridine and derivatives, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-(3-[vinylsulfonyl]phenyl)-naphthalimide-3,6-disulfonate dilithium salt, N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BODIPY, Brilliant Yellow, coumarin and derivatives, cyanine dyes, cyanosine, 4′,6-diaminidino-2-phenylindole (DAPI), bromopyrogallol red, 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, diethylenetriamine pentaacetate, 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, dansylchloride, 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), eosin and derivatives, erythrosin and derivatives, ethidium, fluorescein, 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein isothiocyanate, X-rhodamine-5-(and 6)-isothiocyanate (QFITC or XRITC), fluorescamine, IR-144 (2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indolium hydroxide, inner salt, compound with n,n-diethylethanamine (1:1), CAS No.: 54849-69-3), 5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethyl benzothiazolium perchlorate (IR140), malachite green isothiocyanate, 4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-phycoerythrin, o-phthaldialdehyde, pyrene, pyrene butyrate, succinimidyl 1-pyrene, butyrate quantum dots, Reactive Red 4 (Cibacron Brilliant Red 3B-A), rhodamine and derivatives, 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, Cyanine-3 (Cy3), Cyanine-5 (Cy5), Cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7), IRD 700, IRD 800, Alexa 647, La Jolta Blue, phthalo cyanine, and naphthalo cyanine.
Such detectable labels relevant for nucleic acid molecular probes include those appropriate for quantitative PCR assay detection methods such as a TAQman, or SYBR green assay, for example, without limitation, tetrachlorofluorescein, and 6-carboxyfluorescien, along with quenchers such as tetramethylrhodamine.
The term immunohistochemistry or IHC as used herein refers to another desirable method for use to determine a biomarker expression level. It refers to common diagnostic tool utilising specific molecular probes, most often antibodies, to accurately identify expression of structures with reference to tissue morphology, usually a protein. The method often comprises multiple steps, applying primary antibodies (antigen, or biomarker specific), which may be labelled with a detectable label, or followed by secondary ligands, or antibodies bearing detectable labels, most often enzymes which may act on a substrate to form a chromagen which may be visualised by microscopy. To identify tissue structures and cells, a counter stain of a different colour (e.g. haematoxylin) is often applied following chromagen development.
Such detectable labels include those appropriate for molecular probes for immunohistochemistry detection methods, for example, without limitation, streptavidin or avidin (amplified with biotin), or a chromogenic reporter enzyme such as alkaline phosphatase (substrates: Fast Red, nitro blue tetrazolium), or horseradish peroxidase (substrates: 3,3′ diaminobenzidine, aminoethyl carbazole).
Another particularly useful means of analysis of biomarker expression encompassed herein is imaging mass cytometry (IMC). An exemplary workflow of IMC (Giesen et al., Nature Methods, 2014 April; 11(4):417-22) comprises preparation of tissue sections for isotope, or metal-chelated antibody labelling using IHC protocols.
Such detectable labels include those appropriate for molecular probes for mass cytometry detection methods, for example, without limitation, the isotope labels listed in Table. 1. Then, tissue samples are positioned in a laser ablation chamber. The tissue is ablated and transported by a gas stream into a time of flight mass spectrometer, such as a CyTOF (Fluidigm) for mass cytometry analysis. The measured isotope signals are plotted using the coordinates of each single laser shot, and a multidimensional tissue image is generated. Single-cell features and marker expression are determined, allowing the investigation of cell subpopulation properties within the analysed tissue.
The term lymph node metastasis, as used herein refers to a cancer originating in breast tissue, which has spread to a lymph node (LN). The clinical measure of nodal status quantifies the number and/or locations of LNs with metastases in clinical Tumour-Node-Metastasis staging of breast cancer patients (Cserni, G. 2018, Virchows Arch. 472:697). Patients without metastases in the sentinel LNs are assigned to nodal status NO, which is the best prognosis group. Lymph node metastasis encompasses node-positive patients are categorized into levels N1, N2, or N3, which describe increasing numbers of axillary LNs with metastases or increasing distance of involved (non-axillary) LNs from the primary tumour (high node status) and coincide with increasingly worse survival.
The terms patient sample, or lymph node metastasis sample as used herein can refer to tissue obtained from a patient LN determined to comprise cancer cells (and optionally healthy tissue), for example a biopsy, or excised LN. The sample may be in the form of a cell preparation or lysate, a nucleic acid preparation (particularly isolated mRNA or a cDNA derivative), a tissue section, or embedded within a tumour microarray. A sample format the is particularly useful according to certain thresholds provided by the invention is a histology section obtained a LN metastasis sample embedded in a tumour microarray on the same slide as a control healthy tissue sample as a negative control.
As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.
As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).
As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.
A first aspect of the invention provides a method to determine the prognosis of a breast cancer patient, by performing an analysis of breast cancer biomarker expression in a tissue sample obtained from a site of lymph node metastasis. The method is suited to predicted the cancer outcomes, or stratifying patients according to what treatments they might be likely to respond to, in order to achieve better outcomes for breast cancer that has spread, or metastasised to a lymph node.
The method comprises the following steps:
Firstly, contacting a lymph node metastasis sample taken from the patient with a molecular probe conjugated to a detectable label specific for either of the prognostic biomarkers GATA3 or p53. In alternative embodiments, a lymph node metastasis sample is contacted with two molecule probes, one specific for GATA3 and the other for p53, each probe conjugated to distinct detectable labels. In some embodiments where both GATA3 expression, and p53 expression are assessed, two distinguishable molecular probes, one which binds specifically to GATA3, and one which binds specifically to p53, are applied to a single patient sample, for example, a histology slide, tissue lysate, or nucleic acid preparation. In the case of such co-administration of molecular probes to a sample, biomarker expression is then assessed by measuring the amount of distinct detectable labels associated with each molecular probe. In other embodiments, two molecule probes, one which binds specifically to GATA3, and one which binds specifically to p53, are applied to two different patient samples obtained from the patient, for example, consecutive histology slides, or nucleic acid sample replicates.
This step optionally comprises de-paraffinization, or antigen retrieval steps, removing unbound molecular probe after an appropriate interval of exposure to the sample, or amplification of the signal, for example using an enzyme such as a polymerase, or a chromogenic reporter and substrate, or a secondary antibody.
Secondly, the biomarker expression level is determined using a methodology sensitive to amount, or level of the detectable marker present in the sample, or in a portion of the sample.
Thirdly, the statistical significance of the biomarker expression level is established in relationship to a measure of patient prognosis. Statistical significance may be revealed by determining the biomarker expression level in breast cancer patient lymph node sample in a cohort of sufficient size to permit an analysis powered to identify biomarker expression levels relative to patient outcomes, such as survival at an indicated timepoint, as demonstrated in the examples.
Lastly, the patient is assigned a good prognosis or a poor prognosis based on the biomarker expression level. In some embodiments, the expression level of the biomarkers in the cohort may be divided by thresholds associated with significantly different patient outcomes, as the expression of p53, and GATA3 is continuously associated with better survival. In other embodiments, the expression level is compared to a control sample with a known clinical outcome, representing a good or bad prognosis.
In certain embodiments, the higher the GATA3 expression level, the higher the likelihood of good prognosis. In other embodiments, the lower the p53 expression level, the higher the likelihood of good prognosis assigned to the patient. In still other embodiments, the higher the p53 expression level, the higher the likelihood of a poor prognosis assigned. In still other embodiments, the lower the GATA3 expression level, the higher the likelihood of a poor prognosis. In particular embodiments, two predictive biomarker relationships are combined to provide a more accurate measure of prognosis, for example a patient with a sample characterised by low GATA3 expression and high p53 expression may be assigned a high risk of poor prognosis.
Good or bad prognosis according to this aspect of the invention may refer to a patient outcome, such as survival relative to the average outcome of a cohort of breast cancer patients, or may be relative to patients not assigned the good, or bad prognosis, respectively. Values attached to patient prognosis, for example, overall survival, may be considered to be provisional in that ongoing analysis in larger cohorts, or cohorts with different characteristics or co-morbidities may refine these values based on continuing analysis.
In one embodiment of the method wherein only the expression level of GATA3 is determined, a good prognosis of about 65% probability of survival at 300 months can be assigned to a patient with a LN sample with a high GATA3 expression level, or a poor prognosis with about 50% probability of survival at 100 months can be assigned to a patient with a LN sample with a low GATA3 expression level, using the thresholds for expression level as determined in an immunohistochemistry sample in
In another embodiment of the method wherein only the expression level of p53 is determined, a good prognosis of about 60% probability of survival at 300 months can be assigned to a patient with a LN sample with a low p53 expression level, or a poor prognosis with about 45% probability of survival at 100 months can be assigned to a patient with a LN sample with a high p53 expression level, using the thresholds for expression level as determined in an immunohistochemistry sample in
In a particular embodiment of the method both the expression level of GATA3 and p53 are determined, which is optionally associated with assignment of a good prognosis of about 60-65% probability of survival at 300 months to any patient with a LN sample with a high GATA3 expression level and/or low p53 expression level, or a poor prognosis with about 10% probability of survival at 100 months to a patient with a LN sample with a low GATA3 expression level, and/or a high p53 expression level using the thresholds for expression level as determined in an immunohistochemistry sample in
The choice of molecular probe according to this aspect of the invention is not particularly limited, as long as it is specific for, or sensitive to expression of the biomarker genes at either the protein or mRNA level. Similarly, many types of detectable label known in the art are suitable for this purpose, paired with a methodology for determining the biomarker expression level that is sensitive to the amount of detectable marker conjugated to the molecular probe specific for p53 and/or GATA3 mRNA, or protein in the sample. Molecular probes of use for this purpose include, but are not limited to, PCR and real time PCR primers, or mRNA hybridising nucleic acid probes, antibodies, or antibody-like molecules, conjugated to fluorescent markers, isotopes, or chromogenic immunohistochemistry reporters.
In particular embodiments, the method provides the use of an antibody, antibody-like molecule and/or nucleic acid probe as molecular probe, particularly wherein the molecular probe is an antibody. Desirable detectable labels according to this embodiment of the invention include, without limit an immunohistochemistry reporter enzyme, an isotope, and/or a fluorescent dye, particularly an antibody bearing a chromogenic enzyme for detection by a standard immunocytochemistry protocol.
In another embodiment of the method according to the invention, the patient is assigned a good prognosis if the patient sample is characterised by a GATA3 expression level greater than or equal to (≥) a GATA3 threshold, and/or a p53 expression level below (<) a p53 threshold, and/or the patient is assigned a poor prognosis if the patient sample is characterised by a p53 expression level ≥the p53 threshold, and/or a GATA3 expression level <the GATA3 threshold.
Statistically significant thresholds suitable for the purpose may be determined by analysing a cohort of breast cancer lymph node metastasis patients with known outcome by means known in the art, including without limit a defined expression level relative to the background biomarker expression of a control sample, or relative to positive or negative control samples. These may be determined in a sample analysed either in bulk (for example, with qPCR, western blot or average intensity analysis of a detectable label in an immunohistochemistry image), or a for value representative of the sample analysed at the level of a single cell (for example, the number of cells expressing a high level of GATA3 or p53).
Certain embodiments of this aspect of the method according to the invention provide an example of a threshold for use to assign samples analysed in bulk (as opposed to per cell). In other words, the statistical significance of the biomarker expression level is assessed at the level of the whole sample, or a portion of the sample comprising a plurality of cells, and compared to a threshold value, for example derived from a negative control sample analysed previously or concurrently to the sample.
In particular embodiments relevant to measurement of biomarker expression level in a bulk, multicellular assay, for example using antibodies for p53 or GATA3 conjugated to a chromogenic enzyme or isotope, measured by immunohistochemistry, or imaging mass cytometry at the level of biomarker signal intensity across the whole image, the method according to the invention provides the following thresholds:
In one embodiment, negative control expression level is the biomarker expression level determined as specified or a negative control sample, particularly a healthy tissue control sample, for example healthy liver. In some embodiments, the threshold for good prognosis is a GATA3 expression level 100, 150, 200, 250, 300, 350, or 400 times higher expression of GATA3 expression level of a negative control sample.
The data presented in
Another embodiment relates to thresholds distinguishing samples associated with good or poor prognosis based on a determining biomarker expression at the level of single cells. A single cell can be identified by suitable means known in the art, such as forward versus side scatter by flow cytometry, or nuclear staining by a DNA-binding dye such as haematoxylin, Hoechst or DAPI for microscopy. It is understood that the biomarker expression of a plurality of cells must be determined in order to calculate an accurate value representing the proportion of biomarker high cells present in the sample, particularly several hundred or thousands of individual cells. These measurements may be obtained, for example, using antibodies for p53 or GATA3 conjugated to a chromogenic enzyme or isotope, measured by immunohistochemistry, flow cytometry or mass cytometry at the level of biomarker signal intensity per cell as demonstrated in the examples. In similar current clinical practice to assess the biomarker Ki67, a counter stain is used to identify at least 500 individual cells containing nuclei, and positive nuclei are as a proportion of total cells (Dowsett M. 2011 J. Natl. Cancer Inst. 103(22):1656).
According to this embodiment, a cell may be classified high, or positive for p53 or GATA3 expression if the biomarker expression level of the cell is ≥10 times a negative control sample. Those skilled in the art will recognise that such a threshold value for biomarker expression may be adjusted depending on what fold change is required to differentiate from noise/the level of background signal present in a cell from a negative control sample (as specified above) contacted with the same molecular probe and detectable label used to examine the patient sample.
According to this embodiment, good prognosis may be assigned to a patient whose sample is determined to have a GATA3 expression level, in other words a proportion of GATA high cells, at or above a threshold of 20% GATA3 positive cells, and particularly ≥30% GATA3 positive cells, and/or where fewer than 6% of cells are p53 high. Conversely, bad prognosis may be assigned to a patient when a sample has fewer than 20% GATA3 positive cells, and/or at least 6% p53 high cells.
The examples provided herein demonstrate that either p53 or GATA3 expression level alone (
Certain embodiments of the method to predict breast cancer patient outcome relate to use of a means of obtaining information about the expression level of the biomarkers GATA3 and/or p53 that comprises obtaining an image of the ex vivo lymph node metastasis sample, for example, without limits, light microscopy, immunohistochemistry, or mass cytometry used to measure bulk or per cell expression levels.
Other embodiments of the method to predict breast cancer patient outcome according to the invention relate to determining the biomarker expression level in a sample on a histology slide, for example a monolayer of adherent ex vivo lymph node metastasis cells or ex vivo lymph node metastasis cells otherwise immobilised on a solid surface.
Particular embodiments of the method to predict breast cancer patient outcome according to the invention relate to the measurement of biomarker expression level in a sample which retains the original tissue morphology of the sample, encompassing sections of an ex vivo lymph node metastasis sample, for example, a histology section of an excised LN, or a tumour microarray comprising multiple patient samples or controls. A particularly helpful format includes slides bearing an additional healthy liver tissue section embedded under the same conditions alongside the patient sample. Appropriate samples may be obtained by sample preservation methods known in the art, such as cryopreservation, optionally using standard optimal cutting temperature compound, colloidal silica, or paraffin embedding processes.
Further embodiments of the method of particular use in reference to the original tissue morphology sample formats listed above relate to use of a method permitting measurement of the expression level of the biomarkers GATA3 and/or p53 at a cellular, or subcellular level. Measurement methods of particular use for this purpose include imaging mass cytometry at a subcellular resolution, particularly at a resolution of ≤5 μm, or even ≤1 μm, or immunohistochemistry paired with microscopy analysis.
In particular embodiments, the measurement of biomarker expression levels is made using immunohistochemistry methodology paired with microscopy. The data presented in
Another aspect of the invention relates to pharmaceutical compositions comprising a hormone-targeted antineoplastic drug and/or its pharmaceutically acceptable salt, for use in treating breast cancer with LN metastasis in a patient who has been assigned a good prognosis according to the method as specified in the above aspects of the invention, as GATA3 expression was demonstrated to correlate with the presence of hormone expression tumour cells.
In one embodiment, the pharmaceutical composition for use in a patient assigned a good prognosis comprises a selective estrogen receptor modulator (SERM) antineoplastic drug, particularly a SERM drug selected from raloxifene, toremifene or tamoxifen.
In one embodiment, the pharmaceutical composition for use in a patient assigned a good prognosis comprises a selective estrogen receptor degraders (SERD) antineoplastic drug, particularly a SERD antineoplastic drug selected from fulvestrant, brilandestrant and elacestrant; and/or
In one embodiment, the pharmaceutical composition for use in a patient assigned a good prognosis comprises an aromatase inhibitor antineoplastic drug, particularly an aromatase inhibitor antineoplastic drug selected from exemestane, letrozole, vorozole, formestane, fadrozole and anastrozole;
Aggressive cytotoxic cancer treatments may not be required, or desirable, in patients who have been assigned a good prognosis according to the invention, who are likely to respond well to hormone-targeted treatment. Thus, in some embodiments, a pharmaceutical composition comprising a hormone-targeted antineoplastic drug is provided for use in a patient who is not receiving concomitant non-hormone targeted antineoplastic treatment. The term concomitant here refers to a medically relevant window surrounding administration of the specified hormone-targeted antineoplastic drugs. In other words, the patient is not currently receiving, has not recently received, and is not scheduled to receive within a medically relevant window (for example within one to several months) a non-hormone-targeted antineoplastic treatment.
In one embodiment, the pharmaceutical composition comprising a hormone targeting treatment is provided for use in a good prognosis patient not concomitantly receiving a pharmaceutical formulation comprising an alkylating antineoplastic drug, particularly an alkylating antineoplastic drug selected from altretamine, bendamustine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, chlorambucil, dacarbayine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiptepa, or trabectedin;
In one embodiment, the pharmaceutical composition comprising a hormone targeting treatment is provided for use in a good prognosis patient not concomitantly receiving a pharmaceutical formulation comprising an antineoplastic platinum complex drug, particularly an antineoplastic platinum complex drug selected from carboplatin, satraplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, picoplatin, triplatin tetranitrate;
In one embodiment, the pharmaceutical composition comprising a hormone targeting treatment is provided for use in a good prognosis patient not concomitantly receiving a pharmaceutical formulation comprising a mitotic inhibitor-type taxane-type antineoplastic drug, particularly a mitotic inhibitor-type taxane-type antineoplastic drug selected from capazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine, and vinorelbine; and/or
In one embodiment, the pharmaceutical composition comprising a hormone targeting treatment is provided for use in a good prognosis patient not concomitantly receiving a pharmaceutical formulation comprising an anthracycline-type antineoplastic drug, particularly an anthracycline-type antineoplastic drug selected from daunorubicin, doxorubicin, epirubicine, idarubicin, mitoxantrone and pixantrone;
In one embodiment, the pharmaceutical composition comprising a hormone targeting treatment is provided for use in a good prognosis patient not concomitantly receiving radiation treatment.
Conversely, the tumours of patients assigned a poor prognosis according to the method specified above largely lacked hormone receptor expression, suggesting clinical justification for use of an aggressive, systemic anti-neoplastic treatment, or a combination of several treatments, instead of a hormone-targeted therapy. Accordingly, another aspect of the invention provides a pharmaceutical composition comprising at least one non-hormone targeted antineoplastic drug, and/or its pharmaceutically acceptable salt, for use in treatment of cancer of a breast cancer patient assigned a poor prognosis according to the method above.
Certain embodiments of this aspect of the invention relate to administration of one, or two, or even three non-hormone targeted antineoplastic drugs, or treatments, to a breast cancer patient who has been assigned a poor prognosis according to the method as specified above.
In one embodiment, the pharmaceutical composition for use in a patient assigned a poor prognosis comprises an alkylating antineoplastic drug, particularly an alkylating antineoplastic drug selected from altretamine, bendamustine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, chlorambucil, dacarbayine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiptepa, or trabectedin;
In one embodiment, the pharmaceutical composition for use in a patient assigned a poor prognosis comprises an antineoplastic platinum complex drug, particularly an antineoplastic platinum complex drug selected from carboplatin, satraplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, picoplatin, triplatin tetranitrate;
In one embodiment, the pharmaceutical composition for use in a patient assigned a poor prognosis comprises a mitotic inhibitor-type taxane-type antineoplastic drug, particularly a mitotic inhibitor-type taxane-type antineoplastic drug selected from capazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine, and vinorelbine; and/or
In one embodiment, the pharmaceutical composition for use in a patient assigned a poor prognosis comprises an anthracycline-type antineoplastic drug, particularly an anthracycline-type antineoplastic drug selected from daunorubicin, doxorubicin, epirubicine, idarubicin, mitoxantrone and pixantrone;
In some embodiments of this aspect of the invention, one or more non-hormone targeted antineoplastic drugs listed above are provided for use in treating breast cancer in a poor prognosis patient who is receiving, who has recently received, or who is further scheduled to receive a radiation treatment.
A high probability of poor prognosis provides increased certainty, and may promote a clinicians' choice of alternative treatments that may are best suited to extend survival, even if they are unlikely to cure a patient. In certain embodiments, palliative care may be preferred for patients assigned a poor prognosis in the context of the unpleasant side effects caused by many breast cancer treatment regimens or co-morbidities.
Similarly within the scope of the present invention is a method or treating cancer in a patient in need thereof, comprising administering to certain patients a hormone-targeted, or non-hormone targeted antineoplastic drug according to the above description.
The skilled person is aware that any specifically mentioned drug compound mentioned herein may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.
Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.
Topical administration is also within the scope of the advantageous uses of the invention. The skilled artisan is aware of a broad range of possible recipes for providing topical formulations, as exemplified by the content of Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1st Edition, Wiley 2011, ISBN-13: 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2nd Ed., CRC Press 2002, ISBN-13: 978-0824708610); Osborne and Amann (Eds.): Topical Drug Delivery Formulations (1st Ed. CRC Press 1989; ISBN-13: 978-0824781835).
Another aspect of the invention relates to a pharmaceutical composition comprising a hormone-targeted, or non-hormone targeted antineoplastic drug of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.
In certain embodiments of the invention, the antineoplastic drug of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product.
The dosage regimen for the antineoplastic drug of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.
In certain embodiments, the pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.
The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).
The invention further encompasses, as an additional aspect, the use of a hormone-targeted, or non-hormone targeted antineoplastic drug as identified herein, or its pharmaceutically acceptable salt, as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of a condition breast cancer characterized by LN metastasis.
Another aspect of the invention relates to a method of treating a patient having been diagnosed with a breast cancer LN metastasis associated with poor prognosis according to method specified above, comprising administering to the patient an effective amount of at least one non-hormone-targeted antineoplastic drug, particularly a non-hormone-targeted antineoplastic drug as specified above and/or its pharmaceutically acceptable salt. In some embodiments, this method further comprises administering an effective amount of radiation treatment to the patient.
An alternative aspect of the invention relates to a method of treating a patient having been diagnosed with a breast cancer LN metastasis associated with good prognosis according to the method specified above, comprising administering to the patient an effective amount of at least one a hormone-targeted antineoplastic drug and/or its pharmaceutically acceptable salt, particularly a hormone-targeted antineoplastic drug as specified above. In some embodiments, this method specifically does not comprise concomitant administration of an additional non-hormone targeted antineoplastic treatment as specified in the method to treat a patient associated with poor prognosis described above.
The invention further encompasses a kit comprising molecular probes with specificity for biomarkers p53 and GATA3, conjugated to detectable labels as specified herein, for use in an assay to determine biomarker expression levels in a breast cancer patient lymph node metastasis sample.
The invention further encompasses a system for use in characterizing a lymph node metastasis sample obtained from a breast cancer patient, wherein the system comprises molecular probes with specificity for p53 and/or GATA3 such as those listed in Table 1, bearing a detectable label, and a device for analysing the amount of detectable label in a lymph node metastasis sample to obtain a GATA3 and/or p53 expression level. Optionally, the system further comprises classifying or stratifying the breast cancer patient into a group sharing similar cellular features based on the expression level of p53 and/or GATA3.
The invention further encompasses the use of labelled molecular probes, particularly antibodies specific for p53 and/or GATA3 identified herein for use in the manufacture of a kit for the detection of p53 and/or GATA3 in LN metastases tissue derived from breast cancer, optionally associated with a good or bad prognosis.
The method may be embodied by way of a computer-implemented method, particularly wherein the evaluation and the assignment step are executed by a computer. Further, the method may be embodied by way of a computer program, comprising computer program code, that when executed on the computer cause the computer to execute at least the evaluation and/or assignment step. Particularly, the results of the measurement step may be provided to the computer and/or the computer program by way of a user input and/or by providing a computer-readable file comprising information regarding the expression level obtained during the measurement step. Results from the measurement step may be stored for further processing on a memory of the computer, on a non-transitory storage medium.
A further aspect of the invention relates to a data processing apparatus comprising a processer configured to carry out the method according to any one of the aspects of the invention outlined above.
In some embodiments, the assessment of the amount of GATA3, or p53 expression, and optionally, application of a threshold or control to classify a sample, is made with the aid of a computer implemented methods. A microscope, or an imaging mass cytometry machine acquires an image of a lymph node metastasis sample stained with labelled molecular probes such as an antibody specific for said biomarker or biomarkers, for example a GATA3 specific antibody tagged with a detectable label. This image is then provided as input to a computer program configured to identify individual cells within the image, and enumerate the number of cells which are positive for said biomarker. Alternatively, the computer program is configured to measure the amount of biomarker present, i.e. the intensity of marker label signal present, in the whole image acquired of the sample, or of a part of said image. The computer program may be configured to apply a threshold, either an absolute threshold, or based on a matched control sample, to classify the sample based on the biomarker expression to provide a matched patient prognosis.
Wherever alternatives for single separable features such as, for example, data sampling methodologies or medical indication are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a detectable label may be combined with any of the alternative embodiments of data sampling methodology, and these combinations may be combined with any medical indication or diagnostic method mentioned herein.
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
Table 1. list of antibody clones and labels used in this study.
The single-cell phenotypic landscape of primary tumour tissues were compared with disseminated cells in matched LN metastases in a cohort of breast cancer patients matched to clinical and long-term survival information (
Single-cell phenotypes were identified in all 771 primary tumour and 271 metastatic LN tissues based on high-dimensional single-cell marker expression, separated into epithelial and non-epithelial cells and then identified single-cell phenotypes using unsupervised PhenoGraph clustering on each group with a selected set of markers. Within the epithelial cell group, 59 clusters of phenotypically similar tumour cell clusters were identified; most were found at both the primary breast and metastatic LN sites (
To relate disseminated tumour cells to the single-cell phenotypes identified in the primary tumour, primary tumours were compared with matched LN metastases for 203 patients, revealing substantial differences in the tumour cell phenotypes found at the two sites of the same patient. The most abundant cell phenotype cluster differed between primary tumour and LN metastasis in 84% of cases (
Clinically established markers varied greatly between primary tumours and matched LN metastases. Certain hormone receptor-positive cell phenotypes (e.g., epithelial cluster 20) formed the bulk of some primary tumours but were not detected in LN metastases; the disseminated cells of these primary tumours were frequently negative for the hormone receptors (e.g., epithelial cluster 27,
The phenotypic variability between primary tumours and LN metastases was also apparent in correlation analyses between tumour single-cell phenotype clusters in these tissues. Strong positive correlations existed among similar phenotype clusters within primary or LN tissues, as previously observed in primary tumours (Jackson, H. W. 2020 Nature, 578:615). Between matched primary and LN metastatic tissue, however, positive correlations occurred between different phenotypes, including between differing phenotypes characterized by clinically established molecular markers. In accordance with this phenotypic heterogeneity, grouping patients based on either the cluster composition of the primary tumours or that of the LN metastases resulted in substantially different groups. Although the primary tumour groups showed expected enrichments for the clinically assigned molecular subtypes and grades, neither the primary tumour-defined groups nor the LN metastasis-defined groups were associated with nodal status.
Differential abundances of tumour single-cell phenotype clusters were assessed in the primary tumours and matched LN metastases with the goal of identifying tumour cell phenotypes with a propensity to disseminate. Cells with high levels of ER, PR, GATA3, and luminal CKs were more abundant in primary tumours (epithelial clusters 3, 8, 9, 20, 21, 47, 49, 53;
Disseminated Tumour Phenotypes are Associated with Patient Survival
The dissemination of tumour cells to the LNs, reflected by the nodal status, is an important prognostic factor, but current clinical evaluation does not take into account the phenotypes of the disseminated cells. To determine which disseminated tumour phenotypes impact survival and how these phenotypes relate to clinical status, a lasso-regularized Cox proportional-hazards model was applied inputting categorical presence or absence information of all tumour cell phenotypes in the LNs (threshold for presence=5 cells) as well as the clinical classifications of the patients (primary tumour grade, molecular subtype of primary tumour, and nodal status). The model identified a predictive set of disseminating cell phenotypes that stratify the overall survival of node-positive patients; primary tumour grade was the only selected stratifying feature among the clinical classifications (
To illustrate the prognostic value of these predictive single-cell phenotypes, patients with disseminated cells of exclusively bad- or exclusively good-prognosis predictive phenotypes were analysed. Patients with LN metastases containing cells from cluster 29, but not the good-prognosis clusters 9, 23, 34, and 50, had a drastically worse prognosis than those with the opposite pattern (
Intra-Lymph Node Metastasis Heterogeneity is Associated with Patient Survival
Cellular heterogeneity of the metastatic lesion was assessed for association with patient prognosis. Tumour cell heterogeneity was quantified using Shannon entropy and assessed for association with overall patient survival using a Cox proportional-hazards model. Tumor cell heterogeneity was expected to increase the likelihood of the presence of a subclone with metastatic potential and resistant to standard treatment. However, increased intra-lesion heterogeneity in the LN metastases was strongly associated with better patient outcome even when controlled for clinical features such as tumor grade, molecular subtype, and nodal status. When the same analysis was performed on the primary tumors, no such association was observed, and there was also no association between heterogeneity of matched primary tumors and LN metastases. Patterns of association with heterogeneity were observed when modeling primary and disseminated sites independently (594 primary tumors and 213 LN metastases with survival information) and when modeling matched primary and LN metastases simultaneously (166 matched samples with survival information).
To determine whether heterogeneity in LN metastases was correlated with spatial separation or with mixing of phenotypes, the levels of heterogeneity were assessed within spatial communities of neighboring tumor cells (Jackson, H. W. 2020 Nature, 578:615). A mixing score was calculated for each tissue that reflects the average intra-community (i.e., local) heterogeneity. In this scoring system, the low values occur when a single tumor cell phenotype dominates the tissue or when there is no physical contact between different phenotypes, and high values occur when cells of many different phenotypes interact in every community. A strong correlation existed between LN metastasis heterogeneity and mixing of cellular phenotypes (i.e., a high score) and also a significant association of mixing with improved survival (
Features of the Primary Tumour are Associated with Prognostic Disseminating Cells
Correlations between disseminating cell phenotypes associated with poor prognosis and cellular phenotypes in the primary tumours assessed using the binary presence or absence measures of all tumour phenotypes identified. A correlation was observed between the good-prognosis, hormone receptor-positive disseminated cell phenotypes and the presence of luminal and hormone receptor-positive phenotypes in the primary tumour and a correlation between the dangerous p53+ triple-negative disseminated cell cluster and non-luminal cell clusters in the primary tumour tissue (
Next, lasso-regularized logistic regression models identified primary tumour phenotypes predictive of the presence or absence of risk-associated disseminated cell phenotypes. Positive predictors in the primary tumour always included the same cellular phenotype as that of the disseminated cells in question (
p53 and GATA3 Expression in Lymph Node Metastases is Associated with Patient Survival
Multiplexed imaging allows for the identification of complex single-cell phenotypes in cancer tissues but the current clinical practice relies on standard single-stain immunohistochemistry. In order to simplify the identification of prognostic disseminated phenotypes to minimal relevant markers, associations between average marker expression levels in LN metastases and patient survival were examined using a lasso regularized Cox proportional-hazards model with the average disseminated cell expression levels of all markers, in order to identify those that stratify the patients according to overall survival. In accordance with the marker signatures of the prognostic disseminated phenotypes (
To demonstrate that p53 and GATA3 can be used as diagnostic markers with established clinical methods, slides were stained and single-cell marker expression levels quantified in different sections of the same LN metastasis cores using standard immunohistochemistry (
Patient prognosis was observed to increase as GATA3 expression increased, or as less p53 was detected, however the survival of patients below or above particular defined thresholds could also provide distinct patient prognosis. Thresholds for either average biomarker expression levels across all tumour cells of a LN metastasis core, or the proportion of highly expressing cells could effectively discriminate groups with differing patient prognosis (
Survival analysis identified disseminating tumor cell phenotypes associated with distinct prognoses, potentially indicative of different metastatic potential or, alternatively, of different therapeutic responses. For instance, hormone receptor-positive disseminated cells, identified with GATA3 expression by IHC, were associated with good prognosis, and are potentially more sensitive to standard endocrine therapy than other phenotypes. Patients with LN metastases of such lower risk, hormone receptor-positive phenotypes may not need radiation, or aggressive chemotherapy with non-targeted anti-neoplastic drugs in addition to endocrine therapies, and could benefit from studies assessing risks of overtreatment. A non-luminal triple-negative disseminated phenotype characterized by high levels of p53 expression was associated with poor prognosis. These patients are unlikely to benefit from hormone-targeted therapy, and might be advised to enter rapid treatment with aggressive systemic chemotherapy, or combinations of different chemotherapy, perhaps additionally with radiation. Notably, this phenotype was observed in patients with primary tumors spanning all clinically assigned molecular subtypes, showing that this dangerous phenotype is not captured by conventional subtyping of the primary tumor. Risk-associated disseminated phenotypes provided prognostic information beyond that of nodal status alone. Thus, the phenotypes of metastatic cells, frequently distinct from those of the primary tumor, has a strong influence on disease outcome, indicating that the phenotypes of disseminated cells should be considered when treatment decisions are made.
The patient samples and clinical information associated with the breast cancer cohort described in this study were obtained from University Hospital Zurich. Pathologists designed and constructed three tissue microarrays (TMAs) (ZTMA21, ZTMA25 and ZTMA26) of core biopsies from different tissue types of breast cancer patients with a focus on primary tumours and LN metastases, taken at diagnosis and before treatment. The TMAs contain a single 0.6-mm diameter core per patient and available tissue type. Images were obtained of 771 primary tumours, 271 LN metastases, 49 tumour recurrences, 41 distant metastases, and 37 healthy breast tissue samples from 890 patients, resulting in 1245 images. For 263 patients more than one tissue type was available and for 212 patients both a primary tumour and a LN metastasis core were available. For analysis, a minimum threshold of 100 tumour cells was applied per image, in order to exclude cores that may have missed the tumour. As a result, the number of patients used for paired primary tumour and LN metastasis analysis was reduced to 203. This project was approved by the local Commission of Ethics (BASEC-PB-2019-111).
The antibody panel was designed to focus on breast cancer-specific epitopes but also to distinguish epithelial, mesenchymal, endothelial, and the main immune cell types (Tab. 1). Slides were stained as previously described (Shapiro, D. 2017 Nat. Methods: 14:873).
Multiplexed images of the TMA cores were acquired using a Hyperion Imaging System (Fluidigm). A square area around each core of a TMA was acquired at 400 Hz, and the raw data were preprocessed using commercial software (Fluidigm). The cores within a TMA were acquired in a randomized order, and the three TMAs were acquired in one continuous run in order of increasing ZTMA number. In cases of unexpectedly interrupted acquisitions, the remaining part of the core was acquired as a separate image, resulting in few patients with the same core split into two images. Signal spillover between channels was compensated on the single-cell level using the CATALYST R package (v.1.12.1).
The commercial image format was converted to OME-TIFF files, then segmented into single-cells using a combination of ilastik (v.1.3.3, Berg, S. 2019 Nat. Methods 16:e2005970) and CellProfiler (v.3.1.8, McQuin, C. PloS Biol. 201816:e2005970), following the imaginf mass cytmoetry SegmentationPipeline (v0.9). High-dimensional mean marker expressions and spatial single-cell features were extracted using CellProfiler. Cells on the edges of the cores were marked as special cases for spatial analyses, and a circular area of each core was recorded for downstream density calculations. Even with high-quality segmentation, single-cells extracted from images of tissue sections represent tissue slices with potentially overlapping cell fragments. Therefore, the extracted single-cell marker expression can include some information of neighboring cells, especially in densely packed tissues. Cellular neighborhoods were extracted by expanding the circumference of each cell by 4 pixels (4 μm) and recording the overlapping neighbor cell IDs using CellProfiler. The pixel values in the area corresponding to each cell were averaged into mean single-cell marker expressions. The single-cell expression data were normalized between 0 and 1 for each marker using the 99th percentile normalization to account for outliers. All downstream analyses were conducted in R (v.3.6.3). A size threshold was applied to mark extremely small or extremely large cells as potential mis-segmentations and remove them from analyses. Only cores containing a minimum of 100 tumour cells were considered for analysis, in order to exclude potentially misleading cores, which may have missed the tumour bulk. Whenever binary presence or absence of a cell type in a tissue is reported, the threshold for presence was 5 cells rather than 1, in order to increase robustness of the analysis. The “predominant” cell type of a tissue was defined as the most abundant phenotype identified in the core.
To identify single-cell phenotypes, epithelial cells were subjected to unsupervised clustering separately from non-epithelial cells. The R package mclust (v.5.4.6) applied a Gaussian-mixture model to the mean single-cell expressions of panCK, in order to separate panCK+ from panCK− cells. However, certain tumour phenotypes, especially non-luminal triple-negative cells, can have very low expression levels of panCK and may be mistaken for non-epithelial cells by a Gaussian-mixture model. Therefore, an unsupervised clustering step, using the RPhenoGraphimplementation from cytofkit (v.1.10.0, Levine, J. H. Cell 2015 162:184) was used, based on all markers and with a nearest neighbour parameter of 50. This granular clustering step split the single cells into 130 clusters. Each cluster was assigned to the epithelial or non-epithelial subgroup based on the majority assignment of cells by the Gaussian-mixture model, refined when neccesary. The epithelial subclustering was performed using the RPhenoGraph implementation of the cytofkit R package with k=100 for epithelial and k=50 for non-epithelial cells. The higher nearest neighbour parameter for epithelial cell clustering was chosen to balance for the higher number of epithelial markers and large inter-patient variability among tumour cells, which would result in exploding numbers of clusters at low nearest neighbour parameters. Epithelial cell clustering resulted in 59 clusters. The z-scored mean marker levels of the clusters are displayed in
Differential abundance testing was conducted using R functions of the edgeR package (v.3.28.0), inspired by the workflow described and implemented in the R package diffcyt (v.1.6.0). In addition to the tissue type of each sample, the model was provided with the patient IDs, thereby accounting for the paired design of the primary tumours and LN metastases. Normalization factors, calculated via the trimmed mean of M-values method, were included to adjust for composition effects. This analysis was conducted separately on epithelial and non-epithelial cells. A significance threshold of p<0.01 was used to identify the differentially abundant cell types.
The R package survival (v.3.1-12) was used to calculate Kaplan-Meier survival curves and Cox proportional-hazards models. The number of samples used for survival analysis slightly deviated from the described total number of samples because survival information was not available for every patient in the cohort. Occasionally, other missing clinical information also led to the exclusion of some patients from analysis. When the number of predictors to be assessed was too high compared to the number of samples to use a regular Cox proportional-hazards model (e.g., 59 epithelial phenotypes, 213 LN metastasis samples), lasso-regularized Cox proportional-hazards models were calculated using the cv.glmnet function of the R package glmnet (v.3.0-2) with the family “cox” option (Friedman J, J. Stat. Software 2010 33(1):1; Simon N, J. Stat. Software 2011 39(5):1). To increase robustness, the cross validation was run 500 times, and the mean error curves were averaged before choosing the optimal lambda value. The cell-type covariates were based on binary presence or absence values and therefore treated like other categorical variables and not standardized. Hazard ratios of the active covariates of the model at the minimum CV-error are displayed in the figures. The hazard ratios of the lasso selected predictors are displayed without confidence intervals, as p-values and confidence intervals of individual coefficients are invalid after feature selection.
To identify phenotypes in the primary tumour that are predictive of the presence of a disseminated cell type of interest, logistic regression models were applied to predict the binomial presence or absence of the phenotype of interest among the cells in the LN metastasis. Lasso-regularization was used to select the stratifying covariates. The cv.glmnet function of the R package glmnet (v.3.0-2) was used with family “binomial” and 10-fold cross validation. To increase robustness, the cross validation was run 500 times, and the mean error curves were averaged before choosing the optimal lambda value. The cell type predictors were based on binary presence/absence values and therefore treated like other categorical variables and not standardized. The active predictors of the model at the minimum CV-error are displayed in the figures.
Pixel or single-cell level example images shown in the figures were generated using the cytomapper R package. IHC stains on the TMAs were performed at the University Hospital Zurich, with anti-GATA3 and p53 anti bodies and a standard horseradish peroxidase enzymatic detection system, and haematoxylin counterstaining. Using QuPATH, each core in the scanned TMA images was segmented into single-cells, which were classified into tumour and non-tumour cells based on manual training. Average expression levels of tumour cells as well as the fraction of highly expressing tumour cells, defined as 10 fold increased staining intensity over background cell staining obtained from healthy liver samples, were extracted for each core.
The invention further relates to the following items:
| Number | Date | Country | Kind |
|---|---|---|---|
| 21156709.4 | Feb 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/053429 | 2/11/2022 | WO |
