ES2976061A1 - UGT2B10 as a predictor of response to neoadjuvant therapy in patients with HER2-positive and hormone receptor-negative breast cancer - Google Patents

Abstract

The present invention relates to the in vitro use of UGT2B10 expression levels to predict or forecast response to neoadjuvant therapy in the treatment of cancer in patients with HER2-positive, hormone receptor-negative breast cancer. In vitro methods are developed for obtaining useful data and for predicting response to neoadjuvant therapy in this group of patients, as well as kits for carrying out the methods of the invention.

Description

DESCRIPTION

UGT2B10 as a predictor of response to neoadjuvant therapy in patients with HER2-positive and hormone receptor-negative breast cancer

FIELD OF INVENTION

The present invention is within the field of medicine and oncology, and relates to a biomarker for predicting response to neoadjuvant therapy in patients with HER2-positive and hormone receptor-negative breast cancer.

BACKGROUND OF THE INVENTION

Breast cancer is the most common tumor worldwide and the main cause of cancer-related death in women. Its incidence varies according to sex, being 100 times more frequent in women than in men, and according to the geographic region, being more frequent in Europe, Australia and North America. Breast cancer is a heterogeneous disease, whose classical prognostic assessment has been based on clinical and histopathological parameters. The staging of cases has been based primarily on two factors. On the one hand, on lymph node involvement, where relapse-free survival decreases as the number of affected nodes increases, and on the other hand, on tumor size, observing that the smaller the tumor, the greater the disease-free survival. It should be noted that the histological grade or degree of differentiation of the tumor cells is an important prognostic factor for cancer.

Advances in molecular biology techniques have allowed breast tumors to be classified into different molecular subtypes based on differential expression analysis using microarrays. Using these techniques, Perou and Sorlie defined for the first time the following molecular subtypes within breast cancer: luminal A (which represents 25-45% of all breast tumors), luminal B (15-50%), HER2 positive (5-15%) and triple negative (10-20%). Each of these molecular subtypes is associated with differences in both the treatment and prognosis of the cancer (1-3).

In routine clinical practice, a surrogate tumor classification is performed based on immunohistochemical markers (IHC), which have prognostic value: the expression of hormone receptors (HR), these being estrogen receptors (ER) and progesterone receptors (PR), allows to identify patients with a better prognosis and candidates for hormonal therapy; the measurement of the overexpression of the HER2 membrane receptor allows to differentiate a group of more aggressive tumors but candidates for targeted treatment, being also a prognostic marker. On the other hand, the tumor proliferation index, determined by the expression of molecules such as MIB-1 (ki67), has prognostic value since the greater the proliferative activity, the worse the prognosis (4).

For several decades, there have been biological treatments or targeted therapies designed to act precisely on the specific molecular processes that the tumor needs for its growth and progression, thus being more effective and presenting lower toxicity profiles. For example, in HER2-positive breast cancer there are some specific targeted therapies such as the monoclonal antibodies trastuzumab or pertuzumab, among others.

In these patients with HER2-positive breast cancer subtype, neoadjuvant chemotherapy is the treatment of choice for localized or locally advanced non-metastatic breast cancer. A good response to neoadjuvant chemotherapy, such as achieving pathologic complete response or “pCR”, increases the disease-free survival of patients. Although there are various criteria to define pathologic complete response or “pCR”, it is generally defined as the complete disappearance in a pathological analysis of the surgical specimen of the infiltrating component of the tumor, both in the breast and in the armpit. In other words, it is the absence of residual disease in the breast and in the lymph nodes located in the armpit.

Another goal of chemotherapy as a neoadjuvant treatment is to improve surgical options for a patient by converting inoperable tumors into operable ones, which in turn allows for better aesthetic results after surgery.

Therefore, it is of particular interest to try to identify molecular predictors of the response to chemotherapy as neoadjuvant treatment, so that it can be predicted which patients will achieve a pathological complete response or "pCR", or which will have a favorable prognosis, even if they do not achieve a pathological complete response.

DESCRIPTION OF THE INVENTION

The purpose of the present invention is to identify which patients with HER2-positive (HER+) and hormone receptor-negative (HR-) breast cancer will have a real benefit in response to neoadjuvant treatment.

In the present invention, it is identified that the overexpression of the UGT2B10 gene in tumor tissue correlates with the absence of a complete pathological response (pCR) to neoadjuvant therapy in patients with HER2-positive and hormone receptor-negative breast cancer. The invention also relates to a method for predicting the response to neoadjuvant treatment in this type of breast cancer patients, allowing to discern two groups of patients with the same diagnosis but different clinical behavior.

UDP-glucuronosyltransferase (UGT) genes encode enzymes involved in glucuronidation, the process in which UGT enzymes transfer the polar moiety of UDP-glucuronic acid to a wide variety of endogenous compounds, including steroid hormones. They are phase II drug-metabolizing enzymes, with steroid/lipid clearance capacity in the liver and various steroid target tissues. The UGT gene superfamily is divided into UGT1 and UGT2 based on amino acid sequence similarities and target substrates dictated by the specificity of their amino termini. Specifically, UGT1 includes: UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9 and UGT1A10, and UGT2 includes: UGT2A1, UGT2A2, UGT2A3, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17 and UGT2B28. Therefore, the UGT2B10 gene would be within the UGT2B gene family.

As demonstrated in the present invention, quantification of UGT2B10 gene expression in tumor tissue allows patients with a complete pathological response (pCR) to be distinguished from those who will not present one. Pathological complete response (pCR) has been proposed as a prognostic marker of long-term clinical outcomes, such as disease-free survival (DFS) and overall survival (OS). In clinical practice, recognizing patients with a probability of achieving such responses is a challenge for precision medicine, so the identification of new useful biomarkers is vital.

Identifying patients who will not benefit from neoadjuvant therapy would avoid exposing patients to the toxicity, expense and time involved in such treatment, while also enabling them to receive an alternative treatment with a greater chance of success.

Therefore, a first aspect of the invention relates to the use of the UGT2B10 gene as a biomarker to predict or forecast the response to neoadjuvant therapy in the treatment of breast cancer in patients with HER2-positive and hormone receptor-negative breast cancer. Preferably, the neoadjuvant treatment consists of chemotherapy and/or HER2-directed therapy, and more preferably, the HER2-directed therapy consists of the administration of monoclonal antibodies such as trastuzumab and/or pertuzumab.

As derived from the results described below in the embodiments of the invention, overexpression of the UGT2B10 gene in tumor tissue correlates with the absence of complete pathological response to neoadjuvant therapy.

Therefore, a second aspect of the invention relates to an expression profile of the UGT2B10 biomarker to predict or forecast the response to neoadjuvant therapy in the treatment of breast cancer in patients with HER2-positive and hormone receptor-negative breast cancer, where overexpression of the UGT2B10 gene is an indicator of the absence of complete pathological response to neoadjuvant therapy.

In this report, UGT2B10 is defined as the member B10 gene of UDP glucuronosyltransferase family 2 (Homo sapiens), Gene ID: 7365, with accession number NC_000004.12 in NCBI.

In the context of the present invention, UGT2B10 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the UGT2B10 protein, and which would comprise various variants derived from:

a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,

b) nucleic acid molecules whose complementary chain hybridizes with the polynucleotide sequence of a),

c) nucleic acid molecules whose sequence differs from a) and/or b) due to the degeneracy of the genetic code,

d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 2. wherein the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the UGT2B10 protein. Preferably, nucleic acid molecules that are transcribed into the mRNA sequence with access code NM 001075.5. is SEQ ID NO: 1

SEQ ID NO: 1

GGAAAAGAATTATCACATTGCACAAGGATGGCTCTGAAATGGACTACAGTTCTGCTGAT ACAACTCAGTTTTTACTTTAGCTCTGGGAGTTGTGGAAAGGTGCTGGTATGGGCCGCAG AATACAGCCTTTGGATGAATATGAAGACAATCCTGAAAGAACTTGTTCAGAGAGGTCATG AGGTGACTGTACTGGCATCTTCAGCTTCCATTCTTTTTGATCCCAACGACTCATCCACTC TTAAACTTGAAG TTTATCCTACATCTTTAACTAAAACTGAATTTGAGAATATCATCATGCA ATTGGTTAAGAGATTGTCAGAAATTCAAAAAGATACATTTTGGTTACCTTTTTCACAAGAA CAAGAAATCCTGTGGGCAATTAATGACATAATTAGAAACTTCTGTAAAGATGTAGTTTCA AATAAGAAACTTATGAAAAAACTACAAGAGTCAAGATTTGACATCGTTTTTGCAGATGCTT ATTTACCCTGTGGTGAGCTGCTGGCTGAGCTATTTAACATACCCTTTGTGTACAGTCACA GCTTCAGTCCTGGCTACTCATTTGAAAGGCACAGTGGAGGATTTATTTTCCTCCTTCCT ACGTACCTGTTGTTATGTCAAAATTAAGTGATCAAATGACTTTCATGGAGAGGGTAAAAA ATATGCTCTATGTGCTTTTTGACTTTTGGTTCCAAATATTTAATATGAAGAAGTGGGA TCAGTTTTACA GTGAAGTTTTAGGAAGACCCACTACATTATCTGAGACAATGAGGAAAGC TGACATATGGCTTATGCGAAACTCCTGGAATTTTAAATTTCCTCATCCATTCTTACCAAAT GTTGATTTTGTTGGAGGACTCCACTGCAAACCTGCCAAACCCCTACCTAAGGAAATGGA GGAGTTTGTACAGAGCTCTGGAGAAAATGGTGTTGTGGTGTTTTCTCTGGGGTCAATGG TCAGTAACATGACAGAAGAAAGGGCCAACGTAATTGCAACAGCCCTTGCCAAGATCCCA CAAAAGGTTCTTTGGAGATTTGATGGGAATAAACCAGATGCCTTAGGTCTCAATACTCGA CTGTACAAGTGGATACCCCAGAATGACCTTCTAGGTCATCCAAAAACCAGAGCTTTTATA ACTCATGGTGGAGCCAATGGCATCTATGAGGCAATCTACCATGGGATCCCTATGGTGG GCATTCCATTGTTTTTT GATCAACCTGATAATATTGCTCACATGAAGGCCAAGGGAGCAG CTGTTAGAGTGGACTTCAACACAATGTCGAGTACAGACCTGCTGAATGCACTGAAGACA GTAATTAATGATCCTTCATATAAAGAGAATATTATGAAATTATCAAGAATTCAACATGATC AACCAGTGAAGCCCCTGGATCGAGCAGTCTTCTGGATTGAATTTGTCATGCGCCACAAA GGAGCCAAACATCTTCGAGTTGCAGCCCACAACCTCACCTGGTTCCAGTACCACTCTTT GGATGTGATTGGGTTCCTGCTGGCTTGTGTGGCAACCGTGCTATTTATCATCACAAAGT GTTGTCTGTTTTGTTTCTGGAAGTTTGCTAGAAAAGGAAAGAAGGGAAAAAGGGATTAGT TATATCTGAGATTTGAAGCTGGAAAACCTGATAGATAGGAATACTTCAGTTGATTCCAGC AATAAATATT GTGATGCAAGATTTCTTTCTTCCTGTGACAAAAAAAAAATCCTTTCGAAGTC TACCTTGTCAAGTAAAAATTTGTTTTTCAGAGATTTACCACCCAGTTAATGGTTAGAAATA TTCTGTGGCAATGAAGAAAACACTAGGGGAAATAAAAAATAATATAAAGCCATATGAGCT TGTATTGAAATTTGTTGCACTTATATTGAAATGTGATCATGGCTCACTTCAGCCTCAACTT ACTAAGCTCAAGAGGTTCTCTCACCTCAGCCCCCCAAGTAGCTGGGACCATAGGTGCAT GTCACCATGTCCAACTAATTTTTTATTTTTTGTAGTGATGAGATCTCATTGTGTTCTCCAT GCTGATTTCAAACTCCTGGGCTCAAACAATCCTCCCATTTTAGCATCCCAAAGGGATGA GATTACAGGTATGTACCACCATAACTTTACAAAATGAGATTTTTATAATAAGAATGATTCAA ATGTTCAGGGATGAAA GAGTCACTAACATAAAAGAAGAATGGGATGAGGTGAGAAGGAT GAATACAAAAATAATTAGATATTCTTGAAATCAGAAATGTGCTCCCTAATTATATGAAATG TTGTTTGATTACATAAAATAAAGTGGAAATGAATGATTGACTGAACAGCCCACAAGAAGA ATCACTTAATGCTCTGAAAATTACCAGTAAACTGATTAAAATCTAAAATTGCTTTCTGTTA AAGCTTTACTGATTAGTTTTTCTTCCAAAGCTCTCTTGTTTCTAGTTGTTTTCTTGGTCTTA ACTACCCATTATATGCTTTGTTAAAGTGTTTATGCCCTGATTCAATGTGATTATCTCAATT TTTATTTCATTCTGTCCTAACTCTTGCAACCTGCATGTCCTCTTTATTATTGATCAATCCA ACTGCAAAGTTCACCTTACCTGACTAAGGATTATTCATTAAGTTTTACTTGTTTATCTGAC ATTTATTATTTTG TCTCTTTGCTAGTCACTCTGAGCCATGGTCATGATGACTTAGGATTCT GGATCTCTTATGAATAACAAATTTATCCTTAATAAAGTCTCTATACT

SEQ ID NO: 2 MALKWTTVLLIQLSFYFSSGSCGKVLVWAAEYSLWMNMKTILKELVQRGHEVTVLASSASIL FDPNDSSTLKLEVYPTSLTKTEFENNMQLVKRLSEIQKDTFWLPFSQEQEILWAINDMRNFCK DVVSNKKLMKKLQESRFDIVFADAYLPCGELLAELFNIPFVYSHSFSPGYSFERHSGGFIFPP SYV PVVMSKLSDQMTFMERVKNMLYVLYFDFWFQIFNMKKWDQFYSEVLGRPTTLSETMR KADIWLMRNSWNFKFPHPFLPNVDFVGGLHCKPAKPLPKEMEEFVQSSGENGVVVFSLGS MVSNMTEERANVIATALAKIPQKVLWRFDGNKPDALGLNTRLYKWIPQNDLLGHPKTRAFIT HGGANGIYEAIYHGIPMVGIPLFFDQPDNIAHMKAKGAAVRVDFNTMSSTDLLNALKTVINDP SYKENIMKLSRIQHDQPVKPLDRAVFWIEFVMRHKGAKHLRVAAHNLTWFQYHSLDVIGFLL ACVATVLFIITKCCLFFCFWKFARKGKKGKRD

Methods of the invention

Another aspect of the invention relates to an in vitro method of obtaining data useful for predicting or forecasting the response to neoadjuvant therapy in the treatment of cancer in patients with HER2-positive and hormone receptor-negative breast cancer, hereinafter the first method of the invention, comprising:

a) determine the expression levels of UGT2B10 in a sample previously isolated from a patient, and

b) compare the UGT2B10 expression values obtained in a) with a reference quantity.

Preferably, neoadjuvant therapy consists of chemotherapy and/or HER2-targeted therapy, and more preferably HER2-targeted therapy consists of the administration of trastuzumab and/or pertuzumab.

Another aspect of the invention relates to an in vitro method for predicting or forecasting the response to neoadjuvant therapy in the treatment of cancer in patients with HER2-positive and hormone receptor-negative breast cancer, hereinafter the second method of the invention, comprising steps (a) - (b) according to the first method of the invention, and further comprising:

c) assign the individual who presents in an analysis the levels of expression of UGT2B10 overexpressed with respect to the average values of a normal individual, to the group of patients who do not present a complete pathological response to neoadjuvant therapy.

Preferably, neoadjuvant therapy consists of chemotherapy and/or HER2-targeted therapy, and more preferably HER2-targeted therapy consists of the administration of trastuzumab and/or pertuzumab.

A "biological sample" as defined here is a small part of a subject, representative of the whole, and may consist of a biopsy or a sample of body fluid.

Preferably, the biological sample is a tissue sample from the patient's solid breast tumor and/or lymph nodes.

The method of the present invention can be applied to samples from individuals of either sex, i.e., men or women, and at any age. It is preferably applied to samples obtained from women.

In the present invention, "prognosis" is understood as the expected course of a disease and refers to the assessment of the probability according to which a subject suffers from a disease, as well as the assessment of its onset, stage of development, evolution, or regression, and/or the prognosis of the course of the disease in the future. As will be understood by those skilled in the art, such an assessment, although preferred, may not normally be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant portion of the subjects can be identified as having the disease or having a predisposition to it. Whether a portion is statistically significant can be readily determined by the skilled person using various well-known statistical evaluation tools, for example, determination of confidence intervals, determination of significance P values, Student's test or Fisher's discriminant functions, non-parametric Mann Whitney measures, Spearman correlation, logistic regression, linear regression, area under the ROC curve (AUC). Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. Preferably, the p-value is less than 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the present invention enables the disease to be correctly detected differentially in at least 60%, more preferably in at least 70%, much more preferably in at least 80%, or even much more preferably in at least 90% of the subjects in a given group or population analysed.

The first method of the invention involves comparing the expression levels of UGT2B10 with the levels of UGT2B10 of a reference sample or with a median value. In the context of the present invention, "reference sample" is understood as the sample that is used to determine the variation of the expression levels of UGT2B10 of the present invention. In a preferred embodiment, the reference value is obtained from the expression values obtained from a sample with individuals responding to treatment with neoadjuvant therapy in the treatment of cancer in patients with HER2-positive and hormone receptor-negative breast cancer.

Preferably, reference samples are taken from several individuals and combined, such that the reference value reflects the average value of such molecules in the population of individuals responding to neoadjuvant treatment. "Reference value" is the expression level of UGT2B10 in a reference sample. The expression profile in the reference sample may preferably be generated from a population of two or more individuals. The population may, for example, contain 3, 4, 5, 10, 15, 20, 30, 40, 50 or more individuals.

The detection of the amount of UGT2B10 expression product can be carried out by any means known in the state of the art.

Expression levels will give a certain “expression profile”. The term “expression level”, also called “product quantity” or “quantity of expression product” refers to the biochemical material, specifically mRNA.

The measurement of the amount or concentration of the expression product, preferably in a semi-quantitative or quantitative manner, can be carried out directly or indirectly. Direct measurement refers to the measurement of the amount or concentration of the expression product, which is directly correlated with the number of RNA molecules. Such a signal (which can also be referred to as an intensity signal) can be obtained, for example, by measuring an intensity value of a chemical or physical property of said products. Indirect measurement includes the measurement obtained from a secondary component or a biological measurement system (for example the measurement of cellular responses, ligands, "labels" or enzymatic reaction products).

The term "quantity" as used in the description refers to, but is not limited to, the absolute or relative quantity of the expression products, as well as any other value or parameter related to them or that can be derived from them. Such values or parameters include signal intensity values obtained from any of the physical or chemical properties of said expression products obtained by direct measurement. Additionally, such values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.

The term "comparison" as used in the description refers to, but is not limited to, the comparison of the amount of UGT2B10 expression products in the biological sample to be analyzed, also called the test biological sample, with an amount of UGT2B10 expression products in one or more reference samples. The reference sample may be analyzed, for example, simultaneously or consecutively, together with the test biological sample.

In a preferred embodiment of this aspect of the invention, the result may be obtained by any of the following techniques:

(i) an mRNA profiling method, such as a microarray, and/or

(ii) a method comprising PCR (polymerase chain reaction), such as real-time PCR; and/or

(iii) Northern blot, and/or

(iv) immunoassay.

In the invention, the method for determining the result, i.e., the expression level of UGT2B10, does not need to be particularly limited.

Real-time quantitative PCR (polymerase chain reaction) (usually abbreviated as RQ-PCR, RT-qPCR, rt-PCR or qPCR) is a sensitive and reproducible mRNA expression quantification technique that can be particularly used to profile mRNA expression in cells and tissues. Any method can be used to evaluate the results of RT-PCR, and the ACt method and the AACt method may be preferred. The AACt method is described in detail by Livak et al. (Methods 2001, 25: 402-408). (Ct = Cycle threshold values). In practicing the present invention, the AACt method described by Livak et al. (Methods 2001, 25: 402-408) will preferably be used. The AACtmethod will include a 'control sample' and a 'subject sample'. The 'subject sample' is a sample from the subject to be analyzed. Typically, several replicates for each diluted concentration are used to derive the amplification efficiency. PCR amplification efficiency can be defined as percentage amplification (from 0 to 1). During the qPCR reaction, a software typically measures for each sample the cycle number at which fluorescence (indicator of PCR amplification) crosses an arbitrary line, the threshold. This crossing point is the Ct value.

A microarray is an array on a solid substrate (usually a glass slide or a silicon thin film cell) that analyzes large amounts of biological material, in this case a large amount of mRNA or, preferably, its reverse DNA transcripts, which are detectable by specific probes immobilized on the solid substrate.

A Northern blot involves the use of electrophoresis to separate RNA samples by size and subsequent detection with a hybridization probe complementary to (part of) the target RNA sequence of interest.

The term "immunoassay" as used in the present description refers to any analytical technique that is based on the conjugation reaction of an antibody with an antigen. Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunosorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, x-map or protein chips. In another preferred embodiment, the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay). The ELISA is based on the premise that an immunoreactive (antigen or antibody) can be immobilized on a solid support, then putting that system in contact with a fluid phase containing the complementary reagent that can bind to a marker compound. There are different types of ELISA: direct ELISA, indirect ELISA or sandwich ELISA. The term "marker compound", as used in the present description, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and/or chemiluminescent signal that allows the detection and quantification of the amount of antibodies against UGT2B10. The marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and/or quantified directly. This marker compound can bind to the antibody directly, or through another compound. Some examples of marker compounds that bind directly are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 32P or 35S, fluorochromes such as fluorescein or metallic particles, for their direct detection by colorimetry, autoradiography, fluorimetry, or metallography respectively.

In the method of the present invention, the expression of the mRNA can be normalized, preferably relative to the expression of another RNA molecule. Normalization methods are well known in the art.

Once the expression levels in relation to the reference values have been determined, it is necessary to identify whether there are alterations in expression (increase or decrease in expression). Expression (and levels of gene expression product) is considered to be increased in a sample of the subject matter when the levels of increase relative to the reference sample are at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more. Similarly, expression is considered decreased when its levels decrease with respect to the reference sample by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%), at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., absent).

The invention provides a method for assigning a human subject into one of two groups: group 1, comprising subjects identifiable by the method of the invention and group 2, representing the remaining subjects. Individuals identifiable by the method of the invention would be those where the expression levels of UGT2B10 are overexpressed. This group of patients would be classified as “non-responders” to treatment with neoadjuvant therapy. We can also define this group of patients as a group with no complete pathological response to treatment with neoadjuvant therapy. Finally, this group would be considered as a group not eligible for treatment with neoadjuvant therapy.

Another aspect of the invention relates to an anti-HER2 antibody or drug, or any pharmaceutical composition containing same, optionally including pharmaceutically acceptable excipients or vehicles, for use in the treatment of patients suffering from HER2-positive breast cancer, wherein the patient is a responder patient characterized by a level of UGT2B10 expression lower than a pre-established threshold value.

Kit and uses

Another aspect of the invention relates to a kit or device, hereinafter kit or device of the invention, comprising the elements necessary to quantify the expression level of UGT2B10.

In a preferred embodiment the kit or device of the invention comprises:

(a) means for detecting in a biological sample obtained from the subject the expression levels of UGT2B10.

In a more preferred embodiment the kit or device of the invention further comprises:

(b) means of comparing the level of expression (a) with a reference sample.

In an even more preferred embodiment the kit or device of the invention further comprises:

(c) instructions for a medical professional not to administer neoadjuvant therapy in the treatment of cancer to patients with HER2-positive, hormone receptor-negative breast cancer and who have overexpressed levels of UGT2B10.

In particular embodiments, the kit is selected from (a) a kit suitable for PCR, (b) a kit suitable for Northern Blot, (c) a kit suitable for microarray analysis, and (d) an immunoassay. Two or more of these embodiments may also be combined, such that the kit may comprise, for example, both (a) and (c).

In a preferred embodiment the kit or device comprises at least one or more oligonucleotides capable of hybridizing with UGT2B10.

It is preferred that said oligonucleotide(s) be capable of doing so under stringent conditions. Stringency is a term used in hybridization experiments that reflects the degree of complementarity between the oligonucleotide and the nucleic acid; the higher the stringency, the higher percentage of homology between the probe and the nucleic acid bound to the filter. It is well known to the skilled artisan that temperature and salt concentrations have a direct effect on the results that are obtained. It is recognized that hybridization results are related to the number of degrees below the Tm (melting temperature) of the DNA on which the experiment is performed. Stringent conditions are often defined as a wash with 0.1X SSC (saline-sodium citrate (SSC) buffer) at 65°C. (SSC is usually provided as a 20X stock solution, consisting of 3 M sodium chloride and 300 mM trisodium citrate (adjusted to pH 7.0 with HCl)).

The kit or device of the invention may comprise controls, program instructions and information necessary to carry out any of the methods of the invention.

Another aspect of the invention relates to the use of the kit or device of the invention in the method of the invention in any of its embodiments, although its use is not particularly limited.

Automation of the method of the invention by implementing it in a computer program

Another aspect of the invention relates to a computer program comprising instructions for performing the procedure according to any of the methods of the invention.

In particular, the invention encompasses computer programs arranged on or within a carrier. The carrier may be any entity or device capable of supporting the program. Alternatively, the carrier could be an integrated circuit in which the program is included and which has been adapted to execute, or to be used in the execution of, the corresponding processes.

For example, the programs could be embodied in a storage medium such as a ROM, CD-ROM or semiconductor ROM, a USB memory stick, or a magnetic recording medium such as a floppy disk or hard disk. Alternatively, the programs could be supported by a transmissible carrier signal; for example, this could be an electrical or optical signal that could be carried by electrical or optical cable, by radio, or by any other means.

The invention also extends to computer programs adapted so that any processing means can implement the methods of the invention. The computer programs also encompass cloud applications based on said method.

Other aspects of the invention relate to the readable storage medium and the transmittable signal comprising program instructions necessary for the execution of the inventive method by a computer.

Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical features, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will become apparent partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF FIGURES

Figure 1. Differentially expressed transcripts between patients with pathological complete response (pCR) and those without (non-pCR). Representation showing transcripts that meet the selection criteria of a log fold change greater than 1.5 or less than -1.5 and a p-value = 0.05.

Figure 2. Expression data obtained on the Affymetrix Clariom D pico array microarray.

Figure 3. ROC curve of UGT2B10 gene expression.

DETAILED DESCRIPTION OF THE INVENTION

Patients and biological samples

The present study was conducted in 18 patients who underwent breast cancer surgery. Patients were enrolled at the Virgen del Rocío University Hospital in Seville. Informed consent was obtained from all patients and the Ethics Committee of the hospital approved the study. All patients were treated with chemotherapy plus HER2-targeted therapy with trastuzumab and in some cases with trastuzumab and pertuzumab. The clinical characteristics of the patients are summarized in Table 1.

Table 1. Clinical data of the patients included in the study.

RH: hormone receptors; HER2: overexpression of the membrane receptor HER2; pCR: pathological complete response.

Patients were stratified according to their response to neoadjuvant treatment. Patients who achieved a pathological complete response (pCR) formed the responder group, while the determination of residual disease after analyzing the surgical specimen following the RCB (Residual Cancer Burden) criteria (5) was used as a reason for inclusion in the non-responder group.

RNA isolation and hybridization with Clarion D microarray

Total RNA was extracted using the commercial RecoverAll Total Nucleic Acid Isolation kit (Ambion, Austin, TX, USA) following the manufacturer's instructions. RNA concentration was measured using the NanoDrop ND-1000 spectrophotometer (Nanodrop Tech, DE, USA). A total of 18 samples were labeled and hybridized with the Clariom D pico Array microarray (Affymetrix, Santa Clara, CA, USA) following the manufacturer's instructions. Briefly, double-stranded complementary DNA (cDNA) and complementary RNA (cRNA) were synthesized from 30 ng of RNA, then the biotinylated cDNA was hybridized for 16 h in an Affymetrix GeneChip 645 hybridization oven at 45°C. Microarrays were stained using GeneChip Fluidics Station 450. The chip was then scanned using GeneChip™ 3000 scanner. Affymetrix Clariom D .CEL files with intensity data were normalized to yield probe-level signal expression values and the expression pattern of genes, exons, splice variants and related pathways involved in response to neoadjuvant chemotherapy was analyzed using Transcriptome Analysis Console (TAC) software.

Statistical analysis

Statistical analysis was performed using R software for Windows version 4.1.1 and SPSS version 28. For the analysis of differential expression between responders and non-responders, the Mann-Whitney U test was applied and a ROC curve analysis was performed to determine the cut-off point of expression signal with the highest sensitivity and specificity, as well as the area under the ROC curve (AUC). Statistical significance was established for p-values <0.05.

Identification of differentially expressed transcripts

The Clariom D assay allows for broad and deep transcriptome analysis and biomarker discovery. In this work, we have combined it with the Applied Biosystems™ Transcriptome Analysis Console (TAC 4.0) software. These microarrays allow obtaining information on the expression of coding and non-coding RNA. In this study, and with the aim of identifying those transcripts that showed differential expression between responders and non-responders, the following filters were established: the logarithm of the change in expression (fold change) was greater than 1.5 or less than -1.5 and the p-value was less than 0.05.

The application of the filters revealed a differential expression of 954 transcripts of which 643 were overexpressed and 311 underexpressed (Figure 1).

Figure 2 shows the expression data in UGT2B10, which meets the selection criteria obtained in the Affymetrix Clariom D pico array microarray.

The hybridization signals obtained for each of the analyzed samples are represented by a scatter plot. Statistical differences were calculated by applying the Mann-Whitney U test. In Figure 2 for the UGT2B10 gene we see a significant difference between the median signal of non-responders (4 patients, median 5.05) and responders (14 patients, median 3.94) with a p-value of 0.0712.

On the other hand, in order to determine the diagnostic accuracy of this gene as a predictor of response, a ROC curve of UGT2B10 expression was performed. ROC curve analysis is a statistical method to determine the cut-off point (expression signal) at which the highest sensitivity and specificity are achieved, as well as the discriminatory capacity of the test, that is, the capacity to predict patients who do not respond to neoadjuvant treatment.

The ROC curve is represented in Figure 3. It shows the UGT2B10 gene with a cut-off point of 4.12. The sensitivity of the test reaches 100% and the specificity 64%. The area under the curve (which reflects the discriminatory capacity of the test) is greater than 0.80, which indicates that UGT2B10 has an acceptable capacity to discriminate between patients who respond and non-respond to neoadjuvant treatment of HER2-positive, RH-negative breast cancer.

References

1. Perou CM, Surlie T, Eisen MB, Rijn M Van De, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumors. letters to nature 748. Nature. 2000;533(May):747-52.

2. S0rlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10869-74.

3. memoria2020online [Internet]. [cited 2021 Aug 26]. Available from: https://www.geicam.org/memoria2020online/mobile/index.html#p=12

4. Yerushalmi R, Woods R, Ravdin PM, Hayes MM, Gelmon KA. Ki67 in breast cancer: prognostic and predictive potential. Lancet Oncol. 2010 Feb 1;11(2):174-83.

5. http://www3.mdanderson.org/app/medcalc/index.cfm?pagename=jsconvert3.

Claims (12)

1. In vitro use of UGT2B10 expression levels as a biomarker to predict or forecast response to neoadjuvant therapy in the treatment of breast cancer in patients with HER2-positive and hormone receptor-negative breast cancer. 2. Use according to the preceding claim where the neoadjuvant therapy consists of chemotherapy and/or targeted therapy. 3. Use according to the preceding claim where the targeted therapy consists of the administration of trastuzumab and/or pertuzumab. 4. A UGT2B10 biomarker expression profile to predict or forecast response to neoadjuvant therapy in the treatment of breast cancer in patients with HER2-positive and hormone receptor-negative breast cancer where overexpression of the UGT2B10 gene is an indicator of absence of complete pathological response. 5. In vitro method for obtaining data useful for predicting or forecasting the response to neoadjuvant therapy in the treatment of cancer in patients with HER2-positive and hormone receptor-negative breast cancer, which includes: a) determine the expression levels of UGT2B10 in a sample previously isolated from a patient, and b) compare the UGT2B10 expression values obtained in a) with a reference quantity. 6. An in vitro method for predicting or forecasting the response to neoadjuvant therapy in the treatment of cancer in patients with HER2-positive and hormone receptor-negative breast cancer, including steps (a) - (b) of the preceding claim and further comprising: c) assign the individual who presents in an analysis the levels of expression of UGT2B10 overexpressed with respect to the average values of a normal individual, to the group of patients who do not present a complete pathological response to neoadjuvant therapy. 7. The method according to any of claims 5 to 6, wherein the biological sample is solid tumor tissue of the breast and/or lymph nodes. 8. The method according to any one of claims 5 to 7, wherein the expression values of UGT2B10 are obtained by: a) an RNA profiling method, such as a microarray, and/or b) a method comprising PCR (polymerase chain reaction), such as real-time PCR; c) Northern blot and/or d) immunoassay. 9. A kit comprising the elements necessary to quantify the level of expression of UGT2B10 in a biological sample obtained from the subject. 10. The kit according to the preceding claim further comprising means for comparing the expression level of UGT2B10 with a reference sample. 11. The kit according to the preceding claim further comprising instructions for a medical professional not to administer neoadjuvant therapy in the treatment of cancer to patients with HER2-positive and hormone receptor-negative breast cancer and who have overexpressed levels of UGT2B10. 12. Use of the kit according to any of claims 9 to 11 to predict or forecast the response to neoadjuvant therapy in the treatment of cancer in patients with HER2-positive and hormone receptor-negative breast cancer in an individual.