WO2014096500A1 - Method for obtaining data useful for the differential diagnosis of lung cancer - Google Patents

Method for obtaining data useful for the differential diagnosis of lung cancer Download PDF

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WO2014096500A1
WO2014096500A1 PCT/ES2013/070910 ES2013070910W WO2014096500A1 WO 2014096500 A1 WO2014096500 A1 WO 2014096500A1 ES 2013070910 W ES2013070910 W ES 2013070910W WO 2014096500 A1 WO2014096500 A1 WO 2014096500A1
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pkp1
krt15
dsg3
lung cancer
expression
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PCT/ES2013/070910
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Spanish (es)
French (fr)
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María Esther FÁREZ VIDAL
Abel SÁNCHEZ-PALENCIA RAMOS
Mercedes GÓMEZ MORALES
José Antonio GÓMEZ CAPILLA
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Universidad De Granada
Servicio Andaluz De Salud
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Publication of WO2014096500A1 publication Critical patent/WO2014096500A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention is within medicine and molecular biology, and refers to a method of obtaining useful data for the differential diagnosis of different types of lung cancer, allowing the establishment of groups of patients.
  • Lung cancer is the leading cause of cancer death worldwide.
  • Non-small cell lung cancer (NSCLC) ( " 85% of all lung cancers) and small cell lung cancer ( from English Small cell lung cancer (SCLC)) ( « 15%).
  • NSCLC non-small cell lung cancer
  • SCLC English Small cell lung cancer
  • Adenocarcinoma makes up 40% of all NSCLC. This type of cancer is usually found in the outermost regions. of the lung There is also a rare form of adenocarcinoma, called bronchoalveolar carcinoma (Bronchioalveolar Carcinoma (BAC)) that is being seen more frequently worldwide. BAC spreads throughout the lung as opposed to other more common types of lung cancer that form unique tumors.
  • BAC Bronchioalveolar Carcinoma
  • NSCLC NSCLC It is fast growing and can appear anywhere in the lung., Lung cancer.
  • Table 1 Types of lung cancer.
  • Immunohistochemistry is a very valuable tool and often used in the differential diagnosis of lung carcinomas. Availability of specific markers of lung tumor would be important for the differential diagnosis of lung or its histological types. The importance of cell-to-cell-binding proteins (including armadillo proteins) in tumor biology is known, but knowledge is limited with respect to specific intercellular adhesion proteins.
  • the contact between epithelial cells is mediated by several types of cell junctions. These junctions are composed of complex aggregations of transmembrane and plaque proteins, and are typically connected to the Cytoskeleton components. Desmosomes are cell-cell complexes that are found mainly in epithelial tissues. In addition to the constitutive proteins of the desmosomal plaque, at least one of the three classic members of the placophyllin family (PKP1 to PKP3) is necessary for the formation of functional desmosomes. Placophilin 1 (PKP1) is a major component of desmosomal plaque that works to recruit intermediate filaments to cell-cell contact sites through interactions with desmoplaquine.
  • Desmosomal cadherins are possible cell adhesion molecules of the cell binding desmosome type by virtue of their homology with the cadherin class of cell adhesion molecules.
  • Two kinds of desmosomal cadherins are known, namely, desmogleins and desmocolins.
  • the cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with intermediate keratin filaments of the cytoskeleton.
  • evidence has accumulated that several binding proteins play important roles in carcinogenesis, tumor invasion, and metastasis.
  • Multiple studies have involved members of the armadillo family of proteins, including placoglobin and ⁇ -catenin in the aberrant regulation of cell adhesion cells that promotes tumor progression.
  • PKPs and other desmosomal proteins in tumor pathology we lack the evidence of a role for PKPs and other desmosomal proteins in tumor pathology.
  • the treatment for lung cancer is based on the type and stage of the cancer. Treatments may include: surgery to remove the tumor, chemotherapy (medications that kill or reduce the size of the tumor) or radiation (x-rays that destroy or damage cancer cells).
  • the examples of the present invention show the immunohistochemical location of the PKP1, KRT15 and DSG3 proteins involved in intercellular adhesions in seventy-five samples of primary lung NSCLC tumors of untreated patients.
  • the staining pattern of these proteins was different between squamous carcinomas and adenocarcinomas.
  • the adenocarcinomas showed no membrane staining.
  • Membrane staining is characteristic of squamous lung carcinomas for the three proteins analyzed. In our study, in squamous cell carcinomas, a relationship was observed between the presence or absence of these proteins in the membrane and the degree of differentiation with a more intense staining in the best differentiated areas in each neoplasm.
  • a first aspect of the invention relates to the use of any of the PKP1, KRT15, DSG3 genes, or any combination thereof, or any of the PKP1, KRT15, DSG3 proteins, or any combination thereof, for differential diagnosis of lung cancer.
  • lung cancer is non-small cell lung cancer, and more preferably it is squamous cell carcinoma.
  • lung cancer is non-small cell lung cancer, and more preferably it is squamous cell carcinoma.
  • Another aspect of the invention relates to a method of obtaining useful data for the differential diagnosis of lung cancer, which comprises: a) obtaining an isolated biological sample comprising cells of an individual, and b) detecting the level of expression of the PKP1, KRT15, DSG3, or any combination thereof, and / or detect the amount of any of the PKP1, KRT15, DSG3 proteins, or any combination thereof, in the sample isolated from (a). c) Compare the expression of the gene or the genes of step (b) with a reference amount.
  • Another aspect of the invention relates to a method of obtaining useful data for the differential diagnosis of lung cancer, which comprises: a) obtaining an isolated biological sample comprising cells of an individual, and b) simultaneously detecting the level of expression of PKP1, KRT15, DSG3, and / or detect the amount of PKP1, KRT15, DSG3 proteins in the sample isolated from (a). c) Compare the expression of the genes of step (b) with a reference amount.
  • steps (b) and / or (c) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the quantity in step (b) or the computerized comparison in step (c).
  • Another aspect of the invention relates to a method for differential diagnosis of lung cancer in an individual, hereafter referred to as the second method of the invention, which comprises steps (a) - (c) according to the first method of the invention. , and also includes: (d) diagnosing the individual in step (a) as an individual with squamous carcinomas, when he has an increased expression of the PKP1, KRT15 and / or DSG3 gene or a larger amount of the PKP1, KRT15 and / or protein DSG3 in the sample obtained in (a), in relation to the amount of expression detected for said gene or said protein in a population of reference patients.
  • increased expression of the PKP1 gene, or a larger amount of the PKP1 protein is detected in the cytoplasm and cell membrane.
  • the individual of step (a) is diagnosed as an individual with lung adenocarcinoma, when he presents the PKP1 and / or KRT15 protein in the nucleus of the tumor cell. Even more preferably, the Individual of step (a) is diagnosed as an individual with adenocarcinoma, when he does not present the DSG3, PKP1 or KRT15 protein in the membrane, nor in the cell.
  • PKP1 and KRT15 were generally restricted to squamous cell carcinoma and was located in the cytoplasm and mainly in the membrane. The expression of these proteins was more extensive and more intense, especially in well-moderately differentiated carcinoma membranes.
  • the staining pattern of DSG3 was also different between squamous cell carcinomas and adenocarcinomas. Thus, membrane staining was characteristic of squamous cell carcinomas for all three proteins.
  • isolated biological sample includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art.
  • the isolated biological sample of an individual from step (a) is a tissue sample, preferably comprising tumor cells.
  • the detection of gene expression, or the detection of the amount of protein can be performed by any means known in the state of the art.
  • the concentration measurement preferably quantitatively, can be carried out directly or indirectly.
  • Direct measurement refers to the measure of gene expression, based on a signal that is obtained directly from the transcripts of said genes, or from the proteins to which they are translated, and that is directly correlated with the number of molecules of RNA or proteins produced by genes. That signal - to which We can also refer to it as an intensity signal - it can be obtained, for example, by measuring an intensity value of a chemical or physical property of such products.
  • the indirect measurement includes the measurement obtained from a secondary component or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).
  • comparison refers to, but is not limited to, the comparison of the expression levels of the PKP1, KRT15 and / or DSG3 genes in the biological sample to be analyzed, also called the sample.
  • biological problem with the expression levels of the PKP1, KRT15 and / or DSG3 genes of one or several desirable reference samples described elsewhere in the present description.
  • the reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample.
  • the comparison described in section (c) of the method of the present invention can be performed manually or assisted by a computer.
  • Gene expression profile means the gene profile obtained after quantification of mRNA and / or protein produced by the genes of interest or biomarkers, that is, by the genes PKP1, KRT15 and / or DSG3, in a biological sample isolated.
  • the expression profile of the genes is preferably performed by determining the level of mRNA derived from its transcription, after extracting the total RNA present in the isolated biological sample, which can be performed by protocols known in the state of the art.
  • the level of mRNA derived from the transcription of the PKP1, KRT15 and / or DSG3 genes can be determined, for example, but not limited to, by amplification by polymerase chain reaction (PCR), back transcription in combination with the reaction polymerase chain (RT-PCR), quantitative RT-PCR, back transcription in combination with the ligase chain reaction (RT-LCR), or any other nucleic acid amplification method; serial analysis of gene expression (SAGE, SuperSAGE); DNA chips made with oligonucleotides deposited by any mechanism; DNA microarrays made with oligonucleotides synthesized in situ by photolithography or by any other mechanism; in situ hybridization using specific probes labeled with any method of marking; by electrophoresis gels; by membrane transfer and hybridization with a specific probe; by nuclear magnetic resonance or any other diagnostic imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with antibodies or by any other means.
  • PCR polymerase chain reaction
  • the gene expression profile could also be obtained by the detection and / or quantification of the proteins resulting from the translation of the mRNA derived from the transcription of the PKP1, KRT15 and / or DSG3 genes, for example, but not limited to, western immunodetection blot Quantitative detection of the expression of the PKP1, KRT15 and / or DSG3 genes can be performed more preferably by real-time PCR (RT-PCR or RTqPCR).
  • the real-time detection of the amplified products can be carried out by means of the use of fluorescent molecules that are intercalated in the double-stranded DNA or by hybridization with different types of probes.
  • the detection of expression levels of the PKP1, KRT15 and / or DSG3 genes is performed by Q-RT-PCR.
  • Quantitative real-time PCR is a technique for quantifying sensitive and reproducible gene expression that can be used in particular for the expression of the gene profile in cells and tissues. Any procedure may be used for the evaluation of the results of the RT-PCR and the AACt procedure may be preferred.
  • the AACt process should preferably be used as described by Livak et al. ⁇ Methods 2001, 25: 402-408).
  • the AACt procedure will involve a "control sample” and a "subject sample”.
  • the "subject sample” is a sample from the subject to be analyzed.
  • a target gene here: the gene of interest
  • an endogenous control gene as described below
  • the efficiency of PCR amplification can be defined as the percentage of amplification (from 0 to 1).
  • software normally measures the number of cycles of each sample in which the fluorescence crosses an arbitrary line (PCR amplification indicator), the threshold. This crossing point is the Ct value. More diluted samples will cross to subsequent Ct values.
  • the Ct of a nucleic acid from the gene of interest is divided by the Ct of the nucleic acid from the endogenous control in the same sample to normalize the variation in the quantity and quality of RNA between different samples and obtain the relative expression (with respect to the endogenous control) of each of the "sample of the subject" and of the "control sample”.
  • this is carried out in duplicate, triplicate, quadruplicate and similarly, respectively.
  • An ACt value of the control can be adequately obtained by calculating the average of the ACt values obtained from samples of a control group of several individuals with which the values of the "subject sample" are to be compared.
  • the control group (from which the average value is calculated) consists of individuals suitable for the respective purposes (for comparison).
  • the skilled person will learn from this disclosure that a suitable control group is for a specific purpose.
  • the present invention can be practiced by omitting the determination of the ACt value of the control group, that is, determining (only) the ACt value of the "subject sample” and then comparing this with the respective one. average ACt value of the control indicated in the examples.
  • other methods are also available in the state of the art, such as Northern Blot Transfer, or microarrays.
  • the detection of the expression product of the PKP1, KRT15 and / or DSG3 genes is performed by Northern Blot Transfer.
  • the detection of the expression product of the PKP1, KRT15 and / or DSG3 genes is performed by microarrays.
  • the PKP1 gene encodes a member of the placophilin gene family. Placophilin proteins contain numerous armadillo repeats, locates desmosomes and cell nuclei, and participate in the binding of cadherins to the intermediate filaments of the cytoskeleton. This protein may be involved in molecular recruitment and stabilization during desmosome formation. Mutations in this gene have been associated with ectodermal dysplasia / skin fragility syndrome. Two variants of transcription encoding different isoforms have been found for this gene. It is found on chromosome 1 (1 q32).
  • PKP1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 1, and which would comprise various variants from: a) acid molecules nucleic encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, b) nucleic acid molecules whose complementary strand hybrid with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, a
  • the KRT15 gene (keratin 15; K15; CK15; K1 CO) encodes a protein that is a member of the keratin family. Keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins.
  • KRT15 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 3, and which would comprise various variants from: a) acid molecules nucleic encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, b) nucleic acid molecules whose complementary strand hybrid with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 3, and in which the polypeptide encode
  • the DSG3 or Desmoglein 3 (PVA; CDHF6) gene is a transmembrane component glycoprotein of desmosome-binding calcium in vertebrate epithelial cells.
  • PVA Desmoglein 3
  • CDHF6 Desmoglein 3
  • DSG3 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 5, and which would comprise various variants from: a) acid molecules nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 5, b) nucleic acid molecules whose complementary strand hybrid with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 5, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the DSG3 protein.
  • these nucleic acid molecules is the collection in the sequence SEQ ID NO: 6.
  • diagnosis refers to the ability to discriminate between individuals affected or not by squamous cell carcinoma, or affected or not by lung adenocarcinoma.
  • prognosis is understood as the expected evolution 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, developmental status, evolution, or its regression, and / or the prognosis of the course of the disease in the future. As those skilled in the art will understand, such assessment, although preferred, may not be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant part of the subjects can be identified as having the disease or having a predisposition to it.
  • a part is statistically significant, it can be determined by the person skilled in the art using several evaluation tools well-known statistics, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test, etc.
  • Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%.
  • P values are preferably 0.2, 0.1, 0.05.
  • Prediction of the response means, in the context of the present invention, the determination of the probability that the patient responds favorably or unfavorably to a particular therapy or treatment, including surgical treatment.
  • prediction refers to an individual evaluation of any parameter that may be useful in determining the evolution of a patient.
  • the prediction of the clinical response to treatment although it is preferred, does not need to be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant part of the subjects can be identified as having an increased probability of having a positive response.
  • Preferred confidence intervals are at least 50%>, at least 60%>, at least 70%>, at least 80%>, at least 90%), at least 95%>.
  • P values are preferably 0.2, 0.1 or 0.05.
  • the prediction of the clinical response can be made using any assessment criteria used in oncology and known to the person skilled in the art.
  • the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%.
  • the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001.
  • the present invention makes it possible to correctly detect the disease differentially by at least 60%, more preferably at least 70%, much more preferably at least 80%, or even much more preferably at least 90%. of the subjects of a certain group or population analyzed.
  • the reference amount is obtained from the constitutive expression values of the genes, in a group of healthy individuals, or from the expression of the genes in the group of individuals before being subjected to treatment.
  • the reference amount will be, for example, in the case of differentiation between patients affected by lung cancer from healthy individuals, the constitutive expression of the gene or the amount of protein detected in a control group of healthy individuals. However, in the case of subclassification of patients affected by lung adenocarcinoma or those affected by squamous cell carcinoma, the control group will consist of a group of patients with lung adenocarcinoma who did not have that clinical manifestation.
  • the reference amount is obtained from a reference sample.
  • the reference quantity can also be obtained, for example, from the normal distribution limits of an amount found in samples obtained from a population of individuals with lung adenocarcinoma, or squamous carcinoma in different phases, using well-known statistical techniques.
  • the reference sample is obtained from patients before and after treatment.
  • lung cancer is non-small cell lung cancer, and more preferably it is squamous cell carcinoma and adenocarcinomas.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a modulating agent of at least one of the genes that are selected from PKP1, KRT15 and / or DSG3 to treat an individual diagnosed with squamous carcinoma according to the methods of the invention.
  • the modulating agent is an inhibitor of the expression of said genes.
  • Another aspect of the invention relates to an antibody for treating an individual diagnosed with squamous carcinoma, identifiable by a method of the invention, wherein the antibody is selected from among anti-PKP1, anti-KRT15, and / or anti-DSG3.
  • the quantification of the amount of PKP1, KRT15 and / or DSG3 proteins can be done by any of the techniques known to those skilled in the art, preferably by immunological techniques.
  • the immunological techniques are based on precipitation reactions, based on agglutination reactions, immunostaining, radioimmunoassay and radioimmunometric techniques, ELISA ⁇ Enzime Linked ImmunoadSorbent Assay), or any combination thereof.
  • immunological techniques comprise immunomarking.
  • the immunomarking is selected from immunomarking with enzyme conjugated antibodies, immunomarking with fluorochrome conjugated antibodies, or cytometry. Even more preferably, the cytometry is flow cytometry. KIT OR DEVICE OF DIAGNOSIS, MICROARRAY or MICROARRAY OF PROTEINS AND USES
  • kit or device of the invention comprising the elements necessary to analyze the level of expression of the PKP1, KRT15 and / or DSG3 genes.
  • the kit may contain oligonucleotides designed from a known sequence or an mRNA of the genes, and / or capable of hybridizing with the sequence of the PKP1, KRT15 and / or DSG3 genes, for subsequent PCR amplification. More preferably, the sequence of the PKP1, KRT15 and / or DSG3 genes is the nucleotide sequence SEO ID NO: 2, SEO ID NO: 4, and SEO ID NO: 6, respectively.
  • the kit or device of the invention comprises at least one anti-PKP1 antibody, an anti-KRT15 antibody, and / or an anti-DSG3 antibody.
  • the antibody is human, humanized or synthetic.
  • the antibody is monoclonal.
  • the antibody is labeled with a fluorochrome. More preferably, the fluorochrome is selected from the list comprising Fluorescein (FITC), Tetramethylrodamine and derivatives, Phycoerythrin (PE), PerCP, Cy5, Texas, allophycocyanin, or any combination thereof.
  • kit may contain all those reagents necessary to analyze the expression level of the PKP1, KRT15 and / or DSG3 genes by means of any of the methods described earlier in this document.
  • the kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc.
  • the kit can include all the supports and containers necessary for commissioning and optimization.
  • the kit comprises furthermore the instructions for carrying out any of the methods of the invention.
  • kits suitable for RQ-PCR a technique for quantifying sensitive and reproducible gene expression
  • the kit additionally comprises a polyT oligonucleotide primer in addition to the oligonucleotide (s) of the kit.
  • reagents may optionally be included in the kit.
  • a Northern Transfer involves the use of electrophoresis to separate RNA samples by size and subsequent detection with an oligonucleotide (s) (hybridization probe) complementary to (part of) the target sequence of the RNA of interest.
  • s oligonucleotide
  • the kit comprises a microarray, or microarray of the invention.
  • An RNA microarray is a matrix on a solid substrate (usually a glass holder or a cell of a thin silicon film) that evaluates large amounts of different RNAs that are detectable by specific probes immobilized on spots on a solid substrate.
  • Each spot contains a specific nucleic acid sequence, usually a DNA sequence, such as probes (or indicators).
  • the number of spots is not limited in any way, there is a preferred embodiment in which the microarray is customized for the methods of the invention.
  • said custom matrix comprises fifty spots or less, such as thirty spots or less, including twenty spots or less. Therefore, another aspect of the invention relates to a microarray comprising oligonucleotides designed from a known sequence or an mRNA of the genes, and / or capable of hybridizing with the sequence of the PKP1, KRT15 and / or DSG3 genes. More preferably, the sequence of the PKP1, KRT15 and / or DSG3 genes is the nucleotide sequence SEQ ID NO: 2, SEO ID NO: 4, SEO ID NO: 6, respectively.
  • microarray hereinafter microarray of the invention, comprising oligonuleotides or microarrays of single channel designed from a known sequence or an mRNA of the PKP1, KRT15 and / or DSG3 genes. More preferably, the sequence of the PKP1, KRT15 and / or DSG3 genes is the nucleotide sequence SEO ID NO: 2, SEO ID NO: 4, SEO ID NO: 6, SEO ID NO: 8, SEO ID NO: 10, SEO ID NO: 12, SEO ID NO: 14, SEO ID NO: 16 and / or SEO ID NO: 18, respectively.
  • oligonucleotide sequences are constructed on the surface of a chip by sequential elongation of a growing chain with a single nucleotide using photolithography.
  • the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask.
  • the photomask can be physical or virtual.
  • oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 30 nucleotides.
  • oligonucleotides per gene are preferably used.
  • Synthesis in situ on a solid support could be done using ink-jet technology, which requires longer probes.
  • the supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy.
  • the probe is each of the chip samples.
  • the target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc.
  • Another aspect of the invention relates to a protein microarray, hereinafter protein microarray of the invention, comprising anti-PKP1 antibodies, anti-KRT15 antibodies, and / or anti-DSG3 antibodies.
  • the probes are antibodies fixed to glass slides and the targets are samples of serum or tissue.
  • Another aspect of the invention relates to the use of the kit or device, the microarray, or the microarray of the invention, of data collection. useful in the diagnosis of lung adenocarcinoma or squamous carcinoma.
  • Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of any of the methods of the invention (of the first or second method of the invention ).
  • Another aspect of the invention relates to a transmissible signal comprising program instructions capable of having a computer perform the steps of any of the methods of the invention.
  • polynucleotide and “nucleic acid” are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).
  • amino acid sequence peptide
  • oligopeptide oligopeptide
  • polypeptide and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, Chemically or biochemically modified.
  • Fig. 1 Immunohistochemical location of the pKP1 protein in squamous cell carcinoma or squamous cell carcinoma. A) Staining of the membrane. B) Cytoplasm staining. C) Cytoplasm staining with intense staining in the corneal balloon (arrow). D) Intense nuclear staining in cells with more immature appearance. (A, B, E and F 40x; C 10x).
  • Fig. 3 Immunohistochemical location of the KRT15 protein in squamous cell lung carcinoma or epidermoid membranes (40x).
  • the immunohistochemical location of the pKP1, KRT15 and DSG3 proteins was examined in squamous cell carcinoma and lung adenocarcinomas. Seventy-five NSCLC tumors were included in the study, comprising 47 squamous carcinomas and 28 adenocarcinomas. Squamous cell carcinomas were well moderately differentiated in 26 samples and poorly differentiated in 21 samples. The adenocarcinomas were well differentiated moderately in 17 samples and poorly differentiated in 1 1 samples.
  • the immunohistochemical results for the pKP1 protein are summarized in Table 1.
  • the immunohistochemical localization for pKP1 protein was mainly restricted to squamous carcinomas, with a heterogeneous distribution and staining intensity between different carcinomas and in different areas of a carcinoma.
  • the immunohistochemical results for the KRT15 protein are summarized in Table 2.
  • the immunohistochemical location for the KRT15 protein was mainly restricted to squamous carcinomas, with a heterogeneous distribution and staining intensity between different carcinomas and in different areas of a carcinoma. .
  • the immunohistochemical results for the DSG3 protein are summarized in Table 3.
  • N Number of cases Table 3. Analysis of the location of DSG3.

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Abstract

The invention relates to the use of the PKP1, KRT15 and DSG3 genes or the PKP1, KRT15 and DSG3 proteins for the differential diagnosis of lung cancer, to a diagnostic kit and to the uses thereof.

Description

MÉTODO DE OBTENCIÓN DE DATOS ÚTILES PARA EL DIAGNÓSTICO DIFERENCIAL DEL CÁNCER DE PULMÓN  METHOD OF OBTAINING USEFUL DATA FOR THE DIFFERENTIAL DIAGNOSIS OF LUNG CANCER
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se encuentra dentro de la medicina y la biología molecular, y se refiere a un método de obtención de datos útiles para el diagnóstico diferencial de los distinto tipos de cáncer d pulmón, permitiendo el establecimiento de grupos de pacientes. The present invention is within medicine and molecular biology, and refers to a method of obtaining useful data for the differential diagnosis of different types of lung cancer, allowing the establishment of groups of patients.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
El cáncer de pulmón es la principal causa de muerte por cáncer en todo el mundo. Lung cancer is the leading cause of cancer death worldwide.
Los dos tipos principales de cáncer de pulmón son el cáncer de pulmón de células no pequeñas (de inglés Non-small cell lung cáncer (NSCLC)) (« 85% de todos los cánceres de pulmón) y el cáncer de pulmón de células pequeñas (del inglés Small cell lung cáncer (SCLC)) (« 15%). El cáncer de pulmón de células no pequeñas se puede dividir en tres grandes subtipos histológicos: The two main types of lung cancer are non-small cell lung cancer (NSCLC) ( " 85% of all lung cancers) and small cell lung cancer ( from English Small cell lung cancer (SCLC)) ( « 15%). Non-small cell lung cancer can be divided into three large histological subtypes:
- Carcinoma de células escamosas, que supone del 25 al 30% de- Squamous cell carcinoma, which accounts for 25 to 30% of
NSCLC. Este tipo es generalmente encontrado cerca de los bronquios, hacia el centro de la cavidad torácica (del pecho). Es también conocido como carcinoma epidermoide y está usualmente asociado a la exposición al humo de tabaco. NSCLC This type is usually found near the bronchi, toward the center of the thoracic (chest) cavity. It is also known as squamous cell carcinoma and is usually associated with exposure to tobacco smoke.
Adenocarcinoma: conforma el 40% de todos los NSCLC. Este tipo de cáncer se encuentra generalmente en las regiones más externas del pulmón. También existe una forma rara de adenocarcinoma, llamado carcinoma broncoalveolar (Bronchioalveolar Carcinoma (BAC)) que se está viendo con mayor frecuencia a nivel mundial. BAC se disemina a lo largo de todo el pulmón a diferencia de otros tipos de cáncer de pulmón más comunes que forman tumores únicos.Adenocarcinoma: makes up 40% of all NSCLC. This type of cancer is usually found in the outermost regions. of the lung There is also a rare form of adenocarcinoma, called bronchoalveolar carcinoma (Bronchioalveolar Carcinoma (BAC)) that is being seen more frequently worldwide. BAC spreads throughout the lung as opposed to other more common types of lung cancer that form unique tumors.
Se desconoce la causa del BAC. A pesar de presentarse en persona que fuman, generalmente se da en aquellas que nunca han fumado. The cause of BAC is unknown. Despite showing up in person who smoke, it usually occurs in those who have never smoked.
- Carcinoma de Células Grandes: conforma del 10% al 15% de- Large Cell Carcinoma: conforms from 10% to 15% of
NSCLC. Es de crecimiento rápido y puede aparecer en cualquier parte del pulmón., el cáncer de pulmón. NSCLC It is fast growing and can appear anywhere in the lung., Lung cancer.
Figure imgf000004_0001
Figure imgf000004_0001
Tabla 1 . Tipos de cáncer de pulmón. Table 1 . Types of lung cancer.
La capacidad de responder a este problema de salud depende de la continua investigación sobre los mecanismos fundamentales celulares y moleculares que controlan la tumorigénesis y metástasis. The ability to respond to this health problem depends on continuous research on the fundamental cellular and molecular mechanisms that control tumorigenesis and metastasis.
La inmunohistoquímica es una herramienta muy valiosa y de uso frecuente en el diagnóstico diferencial de los carcinomas pulmonares. Disponibilidad de marcadores específicos de tumor de pulmón sería importante para el diagnóstico diferencial de pulmón o de sus tipos histológicos. La importancia de las proteínas de unión de célula a célula-(incluyendo proteínas armadillo) en la biología del tumor se conoce, pero el conocimiento es limitado con respecto a las proteínas específicas de las adhesiones intercelulares. Immunohistochemistry is a very valuable tool and often used in the differential diagnosis of lung carcinomas. Availability of specific markers of lung tumor would be important for the differential diagnosis of lung or its histological types. The importance of cell-to-cell-binding proteins (including armadillo proteins) in tumor biology is known, but knowledge is limited with respect to specific intercellular adhesion proteins.
El contacto entre las células epiteliales está mediado por varios tipos de uniones de células. Estas uniones se componen de complejas agregaciones de proteínas transmembrana y de placa, y están típicamente conectados a los componentes del citoesqueleto. Los desmosomas son complejos célula-célula que se encuentran principalmente en los tejidos epiteliales. Además de las proteínas constitutivas de la placa desmosomal, al menos uno de los tres miembros clásicos de la familia de placofilinas (PKP1 a PKP3) es necesaria para la formación de desmosomas funcionales. La placofilina 1 (PKP1 ) es un componente principal de placa desmosomal que funciona para reclutar filamentos intermedios a los sitios de contacto célula-célula a través de interacciones con desmoplaquina. Las cadherinas desmosómicas son posibles moléculas de adhesión celular del tipo desmosoma de unión celular en virtud de su homología con la clase cadherina de moléculas de adhesión celular. Dos clases de cadherinas desmosómicas son conocidas, a saber, los desmogleínas y la desmocolinas. El dominio citoplasmático de las cadherinas desmosómicas interactuar con las proteínas de placa que a su vez interactúan con filamentos intermedios de queratina del citoesqueleto. En los últimos años, se ha acumulado evidencia de que varias proteínas de unión desempeñan papeles importantes en la carcinogénesis, invasión del tumor, y la metástasis. Múltiples estudios han implicado a miembros de la familia de proteínas armadillo, incluyendo placoglobina y β-catenina en la regulación aberrante de células de adhesión celular que promueve la progresión del tumor. Sin embargo, carecemos de la evidencia de un papel para PKPs y otras proteínas desmosomales en la patología tumoral. The contact between epithelial cells is mediated by several types of cell junctions. These junctions are composed of complex aggregations of transmembrane and plaque proteins, and are typically connected to the Cytoskeleton components. Desmosomes are cell-cell complexes that are found mainly in epithelial tissues. In addition to the constitutive proteins of the desmosomal plaque, at least one of the three classic members of the placophyllin family (PKP1 to PKP3) is necessary for the formation of functional desmosomes. Placophilin 1 (PKP1) is a major component of desmosomal plaque that works to recruit intermediate filaments to cell-cell contact sites through interactions with desmoplaquine. Desmosomal cadherins are possible cell adhesion molecules of the cell binding desmosome type by virtue of their homology with the cadherin class of cell adhesion molecules. Two kinds of desmosomal cadherins are known, namely, desmogleins and desmocolins. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with intermediate keratin filaments of the cytoskeleton. In recent years, evidence has accumulated that several binding proteins play important roles in carcinogenesis, tumor invasion, and metastasis. Multiple studies have involved members of the armadillo family of proteins, including placoglobin and β-catenin in the aberrant regulation of cell adhesion cells that promotes tumor progression. However, we lack the evidence of a role for PKPs and other desmosomal proteins in tumor pathology.
Recientemente (Sanchez-Palencia et al., 2010. Int J Cáncer 129(2):355-364) se han establecido perfiles de expresión génica en NSCLC, en carcinomas primarios de células escamosas o epidermoides y adenocarcinomas. Después del análisis de microarrays de muestras tumorales y no tumorales, el nivel de expresión de 92 genes seleccionados fue validado por qPCR utilizando el test robusto test de Bonferroni en un conjunto independiente de las muestras. En este primer estudio, que mostró resultados acerca de las secuencias de genes expresados diferencialmente en función del tipo de tumor, el estadio y el grado de diferenciación en NSCLC. También puso de manifiesto los datos relacionados con las secuencias de genes expresados diferencialmente correspondientes a las proteínas desmosómicas placofilina 1 , queratina 15 y desmogleína 3 en el cáncer no microcítico de pulmón. Después de que el tipo de cáncer de pulmón es identificado y el estadio determinado, el paciente y su familia pueden discutir opciones de tratamiento con el equipo médico. El tratamiento para cáncer de pulmón se basa en el tipo y estadio del cáncer. Los tratamientos pueden incluir: cirujía para remover el tumor, quimioterapia (medicamentos que matan o reducen el tamaño del tumor) o radiación (rayos X que destruyen o dañan las células cancerosas). Recently (Sanchez-Palencia et al., 2010. Int J Cancer 129 (2): 355-364) gene expression profiles have been established in NSCLC, in primary squamous or squamous cell carcinomas and adenocarcinomas. After microarray analysis of tumor and non-tumor samples, the expression level of 92 selected genes was validated by qPCR using the robust Bonferroni test in an independent set of samples. In this first study, we showed results about gene sequences differentially expressed based on tumor type, stage and degree of differentiation in NSCLC. It also revealed data related to differentially expressed gene sequences corresponding to the desmosomal proteins placofilin 1, keratin 15 and desmoglein 3 in non-small cell lung cancer. After the type of lung cancer is identified and the stage determined, the patient and his family can discuss treatment options with the medical team. The treatment for lung cancer is based on the type and stage of the cancer. Treatments may include: surgery to remove the tumor, chemotherapy (medications that kill or reduce the size of the tumor) or radiation (x-rays that destroy or damage cancer cells).
Es por tanto, necesario, desarrollar un método de diagnóstico diferencial que permita conocer el tipo específico de cáncer de pulmón, así como elestadío en el que se encuentra. It is therefore necessary to develop a differential diagnostic method that allows to know the specific type of lung cancer, as well as the condition in which it is found.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
En los ejemplos de la presente invención se muestra la localización inmunohistoquímica de las proteínas PKP1 , KRT15 y DSG3 involucradas en las adhesiones intercelulares, en setenta y cinco muestras de tumores de NSCLC primarios de pulmón de pacientes no tratados. El patrón de tinción de estas proteínas fue diferente entre los carcinomas escamosos y adenocarcinomas. Los adenocarcinomas no mostraron tinción de membrana. La tinción de membrana es característica de los carcinomas escamosos de pulmón para las tres proteínas analizadas. En nuestro estudio, en los carcinomas de células escamosas, se observó una relación entre la presencia o ausencia de estas proteínas en la membrana y el grado de diferenciación con una tinción más intensa en las áreas mejor diferenciadas en cada neoplasia. La expresión de estas proteínas dibujaron las uniones intercelulares que son características del estrato escamoso del epitelio plano monoestratificado y de neoplasias con este tipo de diferenciación (carcinomas de células escamosas) y se puede utilizar en el diagnóstico de los pacientes afectados por carcinoma de células escamosas del pulmón. Los autores de la presente invención han desarrollado un método que permite el diagnóstico diferencial del cáncer de pulmón mediante la detección de tres biomarcadores, así como un kit de diagnóstico diferencial que permite el establecimiento de grupos de pacientes. The examples of the present invention show the immunohistochemical location of the PKP1, KRT15 and DSG3 proteins involved in intercellular adhesions in seventy-five samples of primary lung NSCLC tumors of untreated patients. The staining pattern of these proteins was different between squamous carcinomas and adenocarcinomas. The adenocarcinomas showed no membrane staining. Membrane staining is characteristic of squamous lung carcinomas for the three proteins analyzed. In our study, in squamous cell carcinomas, a relationship was observed between the presence or absence of these proteins in the membrane and the degree of differentiation with a more intense staining in the best differentiated areas in each neoplasm. The expression of these proteins drew the intercellular junctions that are characteristic of the squamous stratum of the monostratized flat epithelium and of neoplasms with this type of differentiation (squamous cell carcinomas) and can be used in the diagnosis of patients affected by squamous cell carcinoma of the lung. The authors of the present invention have developed a method that allows differential diagnosis of lung cancer by detecting three biomarkers, as well as a differential diagnostic kit that allows the establishment of patient groups.
Por tanto, un primer aspecto de la invención se refiere al uso de cualquiera de los genes PKP1, KRT15, DSG3, o cualquiera de sus combinaciones, o de cualquiera de las proteínas PKP1 , KRT15, DSG3, o cualquiera de sus combinaciones, para el diagnóstico diferencial del cáncer de pulmón. En una realización preferida, el cáncer de pulmón es el cáncer de pulmón de células no pequeñas, y más preferiblemente es el carcinoma de células escamosas. Therefore, a first aspect of the invention relates to the use of any of the PKP1, KRT15, DSG3 genes, or any combination thereof, or any of the PKP1, KRT15, DSG3 proteins, or any combination thereof, for differential diagnosis of lung cancer. In a preferred embodiment, lung cancer is non-small cell lung cancer, and more preferably it is squamous cell carcinoma.
Otro aspecto de la invención se refiere al uso simultáneo de los genes PKP1, KRT15, DSG3, o de cualquiera de las proteínas PKP1 , KRT15, DSG3, para el diagnóstico diferencial del cáncer de pulmón. Sin embargo, el uso independiente de cualquiera de ellos o de cualquiera de sus combinaciones podrían ser suficientes para el diagnóstico, pronóstico o predicción de la respuesta al tratamiento de dicha enfermedad. En una realización preferida, el cáncer de pulmón es el cáncer de pulmón de células no pequeñas, y más preferiblemente es el carcinoma de células escamosas. Another aspect of the invention relates to the simultaneous use of the PKP1, KRT15, DSG3, or any of the PKP1, KRT15, DSG3 proteins, for the differential diagnosis of lung cancer. However, the independent use of any of them or any of their combinations could be sufficient for the diagnosis, prognosis or prediction of the response to the treatment of said disease. In a preferred embodiment, lung cancer is non-small cell lung cancer, and more preferably it is squamous cell carcinoma.
MÉTODO DE OBTENCIÓN DE DATOS ÚTILES Y MÉTODO DE DIAGNÓSTICO DIFERENCIAL METHOD OF OBTAINING USEFUL DATA AND DIFFERENTIAL DIAGNOSTIC METHOD
Otro aspecto de la invención se refiere a un método de obtención de datos útiles para el diagnóstico diferencial del cáncer de pulmón, que comprende: a) obtener una muestra biológica aislada que comprende células de un individuo, y b) detectar el nivel de expresión de los genes PKP1, KRT15, DSG3, o cualquiera de sus combinaciones, y/o detectar la cantidad de cualquiera de las proteínas PKP1 , KRT15, DSG3, o cualquiera de sus combinaciones, en la muestra aislada de (a). c) Comparar la expresión del gen o los genes del paso (b) con una cantidad de referencia. Another aspect of the invention relates to a method of obtaining useful data for the differential diagnosis of lung cancer, which comprises: a) obtaining an isolated biological sample comprising cells of an individual, and b) detecting the level of expression of the PKP1, KRT15, DSG3, or any combination thereof, and / or detect the amount of any of the PKP1, KRT15, DSG3 proteins, or any combination thereof, in the sample isolated from (a). c) Compare the expression of the gene or the genes of step (b) with a reference amount.
Otro aspecto de la invención se refiere a un método de obtención de datos útiles para el diagnóstico diferencial del cáncer de pulmón, que comprende: a) obtener una muestra biológica aislada que comprende células de un individuo, y b) detectar simultáneamente el nivel de expresión de los genes PKP1, KRT15, DSG3, y/o detectar la cantidad de las proteínas PKP1 , KRT15, DSG3, en la muestra aislada de (a). c) Comparar la expresión de los genes del paso (b) con una cantidad de referencia. Another aspect of the invention relates to a method of obtaining useful data for the differential diagnosis of lung cancer, which comprises: a) obtaining an isolated biological sample comprising cells of an individual, and b) simultaneously detecting the level of expression of PKP1, KRT15, DSG3, and / or detect the amount of PKP1, KRT15, DSG3 proteins in the sample isolated from (a). c) Compare the expression of the genes of step (b) with a reference amount.
Los pasos (b) y/o (c) de los métodos descritos anteriormente pueden ser total o parcialmente automatizados, por ejemplo, por medio de un equipo robótico sensor para la detección de la cantidad en el paso (b) o la comparación computerizada en el paso (c). The steps (b) and / or (c) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the quantity in step (b) or the computerized comparison in step (c).
Otro aspecto de la invención se refiere a un método para el diagnóstico diferencial del cáncer de pulmón en un individuo, de ahora en adelante segundo método de la invención, que comprende los pasos (a) - (c) según el primer método de la invención, y además comprende: (d) diagnosticar al individuo del paso (a) como un individuo con carcinomas escamosos, cuando presente una expresión aumentada del gen PKP1, KRT15 y/o DSG3 o una cantidad mayor de la proteína PKP1 , KRT15 y/o DSG3 en la muestra obtenida en (a), en relación a la cantidad de expresión detectada para dicho gen o dicha proteína en una población de pacientes de referencia. En una realización prefrida de este aspecto de la invención, la expresión aumentada del gen PKP1, o una cantidad mayor de la proteína PKP1 se detecta en el citoplasma y la membrana de las células. Another aspect of the invention relates to a method for differential diagnosis of lung cancer in an individual, hereafter referred to as the second method of the invention, which comprises steps (a) - (c) according to the first method of the invention. , and also includes: (d) diagnosing the individual in step (a) as an individual with squamous carcinomas, when he has an increased expression of the PKP1, KRT15 and / or DSG3 gene or a larger amount of the PKP1, KRT15 and / or protein DSG3 in the sample obtained in (a), in relation to the amount of expression detected for said gene or said protein in a population of reference patients. In a preferred embodiment of this aspect of the invention, increased expression of the PKP1 gene, or a larger amount of the PKP1 protein is detected in the cytoplasm and cell membrane.
En otra realización preferida, el individuo del paso (a) se diagnostica como un individuo con adenocarcinoma de pulmón, cuando presenta la proteína PKP1 y/o KRT15 en el núcleo de la célula tumoral. Aún más preferiblemente, el individuo del paso (a) se diagnostica como un individuo con adenocarcinoma, cuando no presenta la proteína DSG3, PKP1 o KRT15 en la membrana, ni de la célula. In another preferred embodiment, the individual of step (a) is diagnosed as an individual with lung adenocarcinoma, when he presents the PKP1 and / or KRT15 protein in the nucleus of the tumor cell. Even more preferably, the Individual of step (a) is diagnosed as an individual with adenocarcinoma, when he does not present the DSG3, PKP1 or KRT15 protein in the membrane, nor in the cell.
En el presente estudio, la expresión de PKP1 y KRT15 estuvo restringida generalmente a carcinoma epidermoide y se localizó en citoplama y principalmente en membrana. La expresión de estas proteínas fue más extensa y más intensa especialmente en membranas de carcinomas bien- moderadamente difenciados. In the present study, the expression of PKP1 and KRT15 was generally restricted to squamous cell carcinoma and was located in the cytoplasm and mainly in the membrane. The expression of these proteins was more extensive and more intense, especially in well-moderately differentiated carcinoma membranes.
El patrón de tinción de DSG3 también fue distinto entre carcinomas epidermoides y adenocarcinomas. Así, la tinción de membrana fue característica de carcinomas epidermoides para las tres proteínas. The staining pattern of DSG3 was also different between squamous cell carcinomas and adenocarcinomas. Thus, membrane staining was characteristic of squamous cell carcinomas for all three proteins.
Nosotros hemos observado una relación en carcinomas epidermoides entre la presencia/ausencia de estas proteínas en la membrana y el grado de diferenciación, encontrando una tinción más intensa en las áreas mejor diferenciadas de los tumores. We have observed a relationship in epidermoid carcinomas between the presence / absence of these proteins in the membrane and the degree of differentiation, finding a more intense staining in the better differentiated areas of the tumors.
Una "muestra biológica aislada" incluye, pero sin limitarnos a, células, tejidos y/o fluidos biológicos de un organismo, obtenidos mediante cualquier método conocido por un experto en la materia. An "isolated biological sample" includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art.
En una realización preferida de este aspecto de la invención la muestra biológica aislada de un individuo del paso (a) es una muestra de tejido, preferiblemente que comprende células tumorales. In a preferred embodiment of this aspect of the invention the isolated biological sample of an individual from step (a) is a tissue sample, preferably comprising tumor cells.
La detección de la expresión de los genes, o la detección de la cantidad de proteína, puede realizarse por cualquier medio conocido en el estado de la técnica. La medida de la concentración, preferiblemente de manera cuantitativa, puede ser llevada a cabo de manera directa o indirecta. La medida directa se refiere a la medida de la expresión de los genes, basada en una señal que se obtiene directamente de los transcritos de dichos genes, o de las proteínas a las que se traducen, y que está correlacionada directamente con el número de moléculas de RNA o de proteínas producidas por los genes. Dicha señal - a la que también podemos referirnos como señal de intensidad - puede obtenerse, por ejemplo, midiendo un valor de intensidad de una propiedad química o física de dichos productos. La medida indirecta incluye la medida obtenida de un componente secundario o un sistema de medida biológica (por ejemplo la medida de respuestas celulares, ligandos, "etiquetas" o productos de reacción enzimática). The detection of gene expression, or the detection of the amount of protein, can be performed by any means known in the state of the art. The concentration measurement, preferably quantitatively, can be carried out directly or indirectly. Direct measurement refers to the measure of gene expression, based on a signal that is obtained directly from the transcripts of said genes, or from the proteins to which they are translated, and that is directly correlated with the number of molecules of RNA or proteins produced by genes. That signal - to which We can also refer to it as an intensity signal - it can be obtained, for example, by measuring an intensity value of a chemical or physical property of such products. The indirect measurement includes the measurement obtained from a secondary component or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).
El término "comparación", tal y como se utiliza en la descripción, se refiere pero no se limita, a la comparación de los niveles de expresión de los genes PKP1, KRT15 y/o DSG3 en la muestra biológica a analizar, también llamada muestra biológica problema, con los niveles de expresión de los genes PKP1, KRT15 y/o DSG3 de una o varias muestras de referencia deseable descrita en otra parte de la presente descripción. La muestra de referencia puede ser analizada, por ejemplo, simultánea o consecutivamente, junto con la muestra biológica problema. La comparación descrita en el apartado (c) del método de la presente invención puede ser realizada manualmente o asistida por ordenador. The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the expression levels of the PKP1, KRT15 and / or DSG3 genes in the biological sample to be analyzed, also called the sample. biological problem, with the expression levels of the PKP1, KRT15 and / or DSG3 genes of one or several desirable reference samples described elsewhere in the present description. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. The comparison described in section (c) of the method of the present invention can be performed manually or assisted by a computer.
Los niveles de expresión de los genes van a dar un determinado perfil de expresión génica. El término "nivel de expresión", también denominado "cantidad producto génico" se refiere al material bioquímico, ya sea ARN o proteína, resultado de la expresión de un gen. Algunas veces se usa una medida de la cantidad de producto génico para inferir qué tan activo es un gen. Se entiende por "perfil de expresión génica" el perfil génico obtenido tras la cuantificación del ARNm y/o de proteína producida por los genes de interés o biomarcadores, es decir, por los genes PKP1, KRT15 y/o DSG3, en una muestra biológica aislada. El perfil de expresión de los genes se realiza, preferiblemente, determinando el nivel de ARNm derivado de su transcripción, previa extracción del ARN total presente en la muestra biológica aislada, lo cual puede realizarse mediante protocolos conocidos en el estado de la técnica. La determinación del nivel de ARNm derivado de la transcripción de los genes PKP1, KRT15 y/o DSG3, puede realizarse, por ejemplo, aunque sin limitarnos, mediante amplificación por reacción en cadena de la polimerasa (PCR), retrotranscripción en combinación con la reacción en cadena de la polimerasa (RT-PCR), RT-PCR cuantitativa, retrotranscripción en combinación con la reacción en cadena de la ligasa (RT-LCR), o cualquier otro método de amplificación de ácidos nucleicos; análisis en serie de la expresión génica (SAGE, SuperSAGE); chips de ADN elaborados con oligonucleótidos depositados por cualquier mecanismo; microarrays de ADN elaborados con oligonucleótidos sintetizados in situ mediante fotolitografía o por cualquier otro mecanismo; hibridación in situ utilizando sondas específicas marcadas con cualquier método de mareaje; mediante geles de electroforesis; mediante transferencia a membrana e hibridación con una sonda específica; mediante resonancia magnética nuclear o cualquier otra técnica de diagnóstico por imagen utilizando nanopartículas paramagnéticas o cualquier otro tipo de nanopartículas detectables funcionalizadas con anticuerpos o por cualquier otro medio. El perfil de expresión génica también podría obtenerse mediante la detección y/o cuantificacion de las proteínas producto de la traducción del ARNm derivado de la transcripción de los genes PKP1, KRT15 y/o DSG3, mediante por ejemplo, pero sin limitarnos, inmunodetección por western blot. La detección cuantitativa de la expresión de los genes PKP1, KRT15 y/o DSG3 puede realizarse más preferiblemente mediante PCR en tiempo real (RT-PCR ó RTqPCR). La detección en tiempo real de los productos amplificados puede llevarse a cabo mediante la utilización de moléculas fluorescentes que se intercalan en el ADN de cadena doble o mediante hibridación con diferentes tipos de sondas. The levels of gene expression will give a certain gene expression profile. The term "expression level", also called "gene product quantity" refers to the biochemical material, either RNA or protein, resulting from the expression of a gene. Sometimes a measure of the amount of gene product is used to infer how active a gene is. "Gene expression profile" means the gene profile obtained after quantification of mRNA and / or protein produced by the genes of interest or biomarkers, that is, by the genes PKP1, KRT15 and / or DSG3, in a biological sample isolated. The expression profile of the genes is preferably performed by determining the level of mRNA derived from its transcription, after extracting the total RNA present in the isolated biological sample, which can be performed by protocols known in the state of the art. The level of mRNA derived from the transcription of the PKP1, KRT15 and / or DSG3 genes can be determined, for example, but not limited to, by amplification by polymerase chain reaction (PCR), back transcription in combination with the reaction polymerase chain (RT-PCR), quantitative RT-PCR, back transcription in combination with the ligase chain reaction (RT-LCR), or any other nucleic acid amplification method; serial analysis of gene expression (SAGE, SuperSAGE); DNA chips made with oligonucleotides deposited by any mechanism; DNA microarrays made with oligonucleotides synthesized in situ by photolithography or by any other mechanism; in situ hybridization using specific probes labeled with any method of marking; by electrophoresis gels; by membrane transfer and hybridization with a specific probe; by nuclear magnetic resonance or any other diagnostic imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with antibodies or by any other means. The gene expression profile could also be obtained by the detection and / or quantification of the proteins resulting from the translation of the mRNA derived from the transcription of the PKP1, KRT15 and / or DSG3 genes, for example, but not limited to, western immunodetection blot Quantitative detection of the expression of the PKP1, KRT15 and / or DSG3 genes can be performed more preferably by real-time PCR (RT-PCR or RTqPCR). The real-time detection of the amplified products can be carried out by means of the use of fluorescent molecules that are intercalated in the double-stranded DNA or by hybridization with different types of probes.
Más preferentemente, la detección de los niveles de expresión de los genes PKP1, KRT15 y/o DSG3 se realiza mediante Q-RT-PCR. More preferably, the detection of expression levels of the PKP1, KRT15 and / or DSG3 genes is performed by Q-RT-PCR.
La PCR cuantitativa en tiempo real (Q-RT-PCR) es una técnica de cuantificacion de la expresión génica sensible y reproducible que se puede usar de manera particular para la expresión del perfil génico en células y tejidos. Se puede usar cualquier procedimiento para la evaluación de los resultados de la RT-PCR y se puede preferir el procedimiento AACt. El procedimiento AACt se describe en detalle en Livak y col. {Methods 2001 , 25:402-408). (Ct = Valores umbral del ciclo). Cuando se lleva la presente invención a la práctica, se deberá usar preferiblemente el procedimiento AACt tal como describen Livak y col. {Methods 2001 , 25:402-408). El procedimiento AACt implicará una "muestra del control" y una "muestra del sujeto". La "muestra del sujeto" es una muestra procedente del sujeto que se va a analizar. Por cada muestra, se incluyen un gen diana (aquí: el gen de interés) y un gen endógeno del control (tal como se describe a continuación) para la amplificación de la PCR a partir de alícuotas (normalmente diluciones en serie). Normalmente se usan varias réplicas de cada concentración diluida para derivar la eficacia de la amplificación. La eficacia de la amplificación de la PCR se puede definir como el porcentaje de amplificación (de 0 a 1 ). Durante la reacción de la qPCR, un software mide normalmente el número de ciclos de cada muestra en el cual la fluorescencia cruza una línea arbitraria (indicadora de la amplificación de la PCR), el umbral. Este punto de cruce es el valor Ct. Muestras más diluidas cruzarán a valores Ct posteriores. Para cuantificar la expresión génica de un gen particular, se divide el Ct de un ácido nucleico procedente del gen de interés por el Ct del ácido nucleico procedente del control endógeno en la misma muestra para normalizar la variación en la cantidad y calidad del ARN entre diferentes muestras y obtener la expresión relativa (con respecto al control endógeno) de cada una de la "muestra del sujeto" y de la "muestra del control". Opcionalmente, esto se lleva a cabo por duplicado, triplicado, cuadriplicado y de manera similar, respectivamente. Se puede obtener de manera adecuada un valor ACt del control calculando el promedio de los valores ACt obtenidos a partir de muestras de un grupo del control de varios individuos con los cuales se van a comparar los valores de la "muestra del sujeto". El grupo del control (del cual se calcula el valor promedio) consiste en los individuos adecuados a los respectivos fines (de comparación). La persona experta aprenderá de esta divulgación que un grupo de control adecuado es para un fin concreto. En una realización particular, la presente invención se puede llevar a la práctica omitiendo la determinación del valor ACt del grupo del control, es decir, determinar (solo) el valor ACt de la "muestra del sujeto" y a continuación comparando posteriormente este con el respectivo valor ACt promedio del control indicado en los ejemplos. Como se ha dicho, otros métodos están también disponibles en el estado de la técnica, como por ejemplo la Transferencia Northern Blot, o los microarrays. Quantitative real-time PCR (Q-RT-PCR) is a technique for quantifying sensitive and reproducible gene expression that can be used in particular for the expression of the gene profile in cells and tissues. Any procedure may be used for the evaluation of the results of the RT-PCR and the AACt procedure may be preferred. The AACt procedure is described in detail in Livak et al. {Methods 2001, 25: 402-408). (Ct = Cycle threshold values). When the present invention is put into practice, the AACt process should preferably be used as described by Livak et al. {Methods 2001, 25: 402-408). The AACt procedure will involve a "control sample" and a "subject sample". The "subject sample" is a sample from the subject to be analyzed. For each sample, a target gene (here: the gene of interest) and an endogenous control gene (as described below) are included for PCR amplification from aliquots (usually serial dilutions). Normally several replicates of each diluted concentration are used to derive the effectiveness of the amplification. The efficiency of PCR amplification can be defined as the percentage of amplification (from 0 to 1). During the qPCR reaction, software normally measures the number of cycles of each sample in which the fluorescence crosses an arbitrary line (PCR amplification indicator), the threshold. This crossing point is the Ct value. More diluted samples will cross to subsequent Ct values. To quantify the gene expression of a particular gene, the Ct of a nucleic acid from the gene of interest is divided by the Ct of the nucleic acid from the endogenous control in the same sample to normalize the variation in the quantity and quality of RNA between different samples and obtain the relative expression (with respect to the endogenous control) of each of the "sample of the subject" and of the "control sample". Optionally, this is carried out in duplicate, triplicate, quadruplicate and similarly, respectively. An ACt value of the control can be adequately obtained by calculating the average of the ACt values obtained from samples of a control group of several individuals with which the values of the "subject sample" are to be compared. The control group (from which the average value is calculated) consists of individuals suitable for the respective purposes (for comparison). The skilled person will learn from this disclosure that a suitable control group is for a specific purpose. In a particular embodiment, the present invention can be practiced by omitting the determination of the ACt value of the control group, that is, determining (only) the ACt value of the "subject sample" and then comparing this with the respective one. average ACt value of the control indicated in the examples. As stated, other methods are also available in the state of the art, such as Northern Blot Transfer, or microarrays.
Por tanto, en otra realización preferida de este aspecto de la invención la detección del producto de expresión de los genes PKP1, KRT15 y/o DSG3 se realiza mediante Transferencia Northern Blot. En otro aspecto de la invención, la detección del producto de expresión de los genes PKP1, KRT15 y/o DSG3 se realiza mediante microarrays. Therefore, in another preferred embodiment of this aspect of the invention the detection of the expression product of the PKP1, KRT15 and / or DSG3 genes is performed by Northern Blot Transfer. In another aspect of the invention, the detection of the expression product of the PKP1, KRT15 and / or DSG3 genes is performed by microarrays.
El gen PKP1, o plakophilin 1 {ectodermal dysplasia/skin fragility syndrome) codifica un miembro de la familia de genes placofilina. Las proteínas placofilina contienen numerosas repeticiones armadillo, localiza a los desmosomas y a los núcleos celulares, y participar en la vinculación de las cadherinas a los filamentos intermedios del citoesqueleto. Esta proteína puede estar implicada en el reclutamiento molecular y la estabilización durante la formación de desmosoma. Las mutaciones en este gen se han asociado con la displasia ectodérmica / síndrome de fragilidad de la piel. Dos variantes de la transcripción que codifican diferentes isoformas se han encontrado para este gen.se encuentra en el cromosoma 1 (1 q32). The PKP1 gene, or plakophilin 1 {ectodermal dysplasia / skin fragility syndrome) encodes a member of the placophilin gene family. Placophilin proteins contain numerous armadillo repeats, locates desmosomes and cell nuclei, and participate in the binding of cadherins to the intermediate filaments of the cytoskeleton. This protein may be involved in molecular recruitment and stabilization during desmosome formation. Mutations in this gene have been associated with ectodermal dysplasia / skin fragility syndrome. Two variants of transcription encoding different isoforms have been found for this gene. It is found on chromosome 1 (1 q32).
En el contexto de la presente invención, PKP1 se define también por una secuencia de nucleótidos o polinucleótido, que constituye la secuencia codificante de la proteína recogida en la SEQ ID NO: 1 , y que comprendería diversas variantes procedentes de: a) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia aminoacídica de la SEQ ID NO: 1 , b) moléculas de ácido nucleico cuya cadena complementaria híbrida con la secuencia polinucleotídica de a), c) moléculas de ácido nucleico cuya secuencia difiere de a) y/o b) debido a la degeneración del código genético, d) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia aminoacídica con una identidad de al menos un 80%, un 90%, unIn the context of the present invention, PKP1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 1, and which would comprise various variants from: a) acid molecules nucleic encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, b) nucleic acid molecules whose complementary strand hybrid with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, a
95%, un 98% o un 99% con la SEQ ID NO: 1 , y en las que el polipéptido codificado por dichos ácidos nucleicos posee la actividad y las características estructurales de la proteína PKP1. Entre dichas moléculas de ácido nucléico se encuentra la recogida en la secuencia SEQ ID NO: 2. El gen KRT15 (keratina 15; K15; CK15; K1 CO) codifica una proteína que es un miembro de la familia de las queratina. Las queratinas son proteínas de filamentos intermedios responsables de la integridad estructural de las células epiteliales y se subdividen en citoqueratinas y las queratinas del pelo. La mayoría de las citoqueratinas de tipo I consisten en proteínas ácidas que están dispuestas en pares de cadenas de queratina heterotípicas y se agrupan en una región en el cromosoma 17 (17q21 .2). En el contexto de la presente invención, KRT15 se define también por una secuencia de nucleótidos o polinucleótido, que constituye la secuencia codificante de la proteína recogida en la SEQ ID NO: 3, y que comprendería diversas variantes procedentes de: a) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia aminoacídica de la SEQ ID NO: 3, b) moléculas de ácido nucleico cuya cadena complementaria híbrida con la secuencia polinucleotídica de a), c) moléculas de ácido nucleico cuya secuencia difiere de a) y/o b) debido a la degeneración del código genético, d) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia aminoacídica con una identidad de al menos un 80%, un 90%, un 95%, un 98% o un 99% con la SEQ ID NO: 3, y en las que el polipéptido codificado por dichos ácidos nucleicos posee la actividad y las características estructurales de la proteína KRT15. Entre dichas moléculas de ácido nucléico se encuentra la recogida en la secuencia SEQ ID NO: 4. 95%, 98% or 99% with SEQ ID NO: 1, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the PKP1 protein. Among said nucleic acid molecules is the collection in the sequence SEQ ID NO: 2. The KRT15 gene (keratin 15; K15; CK15; K1 CO) encodes a protein that is a member of the keratin family. Keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins. Most type I cytokeratins consist of acidic proteins that are arranged in pairs of heterotypic keratin chains and are grouped in a region on chromosome 17 (17q21 .2). In the context of the present invention, KRT15 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 3, and which would comprise various variants from: a) acid molecules nucleic encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, b) nucleic acid molecules whose complementary strand hybrid with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 3, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the KRT15 protein. Among these nucleic acid molecules is the collection in the sequence SEQ ID NO: 4.
El gen DSG3 o Desmogleina 3 (PVA; CDHF6) es una glicoproteína componente del calcio transmembrana de unión de desmosomas en las células epiteliales de vertebrados. Actualmente, tres miembros de la subfamilia antidesmogleína han sido identificados y todos son miembros de la superfamilia de la molécula de adhesión celular cadherina. Estos miembros de la familia de genes antidesmogleína se encuentran en un clúster en el cromosoma 18. Esta proteína se ha identificado como el autoantígeno de la enfermedad pénfigo vulgaris. En el contexto de la presente invención, DSG3 se define también por una secuencia de nucleótidos o polinucleótido, que constituye la secuencia codificante de la proteína recogida en la SEQ ID NO: 5, y que comprendería diversas variantes procedentes de: a) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia aminoacídica de la SEQ ID NO: 5, b) moléculas de ácido nucleico cuya cadena complementaria híbrida con la secuencia polinucleotídica de a), c) moléculas de ácido nucleico cuya secuencia difiere de a) y/o b) debido a la degeneración del código genético, d) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia aminoacídica con una identidad de al menos un 80%, un 90%, un 95%, un 98% o un 99% con la SEQ ID NO: 5, y en las que el polipéptido codificado por dichos ácidos nucleicos posee la actividad y las características estructurales de la proteína DSG3. Entre dichas moléculas de ácido nucléico se encuentra la recogida en la secuencia SEQ ID NO: 6. The DSG3 or Desmoglein 3 (PVA; CDHF6) gene is a transmembrane component glycoprotein of desmosome-binding calcium in vertebrate epithelial cells. Currently, three members of the antidesmoglein subfamily have been identified and all are members of the cadherin cell adhesion molecule superfamily. These members of the family of antidesmoglein genes are found in a cluster on chromosome 18. This protein has been identified as the autoantigen of pemphigus vulgaris disease. In the context of the present invention, DSG3 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 5, and which would comprise various variants from: a) acid molecules nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 5, b) nucleic acid molecules whose complementary strand hybrid with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 5, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the DSG3 protein. Among these nucleic acid molecules is the collection in the sequence SEQ ID NO: 6.
El término "diagnóstico", tal y como se utiliza en la presente invención, se refiere a la capacidad de discriminar entre individuos afectados o no por carcinoma de células escamosas, o afectados o no por adenocarcinoma de pulmón. The term "diagnosis", as used in the present invention, refers to the ability to discriminate between individuals affected or not by squamous cell carcinoma, or affected or not by lung adenocarcinoma.
En la presente invención "pronóstico" se entiende como la evolución esperada de una enfermedad y se refiere a la valoración de la probabilidad según la cual un sujeto padece una enfermedad así como a la valoración de su inicio, estado de desarrollo, evolución, o de su regresión, y/o el pronóstico del curso de la enfermedad en el futuro. Como entenderán los expertos en la materia, tal valoración, aunque se prefiere que sea, normalmente puede no ser correcta para el 100% de los sujetos que se va a diagnosticar. El término, sin embargo, requiere que una parte estadísticamente significativa de los sujetos se pueda identificar como que padecen la enfermedad o que tienen predisposición a la misma. Si una parte es estadísticamente significativa se puede determinar sin más por el experto en la materia usando varias herramientas de evaluación estadística bien conocidas, por ejemplo, determinación de intervalos de confianza, determinación de valores p, prueba t de Student, prueba de Mann- Whitney, etc. Los intervalos de confianza preferidos son al menos el 50%, al menos el 60%, al menos el 70%, al menos el 80%, al menos el 90%, al menos el 95%. Los valores de p son, preferiblemente, 0,2, 0,1 , 0,05. In the present invention "prognosis" is understood as the expected evolution 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, developmental status, evolution, or its regression, and / or the prognosis of the course of the disease in the future. As those skilled in the art will understand, such assessment, although preferred, may not be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant part of the subjects can be identified as having the disease or having a predisposition to it. If a part is statistically significant, it can be determined by the person skilled in the art using several evaluation tools well-known statistics, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test, etc. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. P values are preferably 0.2, 0.1, 0.05.
Por "predicción de la respuesta" se entiende, en el contexto de la presente invención, la determinación de la probabilidad de que el paciente responda de forma favorable o desfavorable a una terapia o a un tratamiento determinado, incluyendo el tratamiento quirúrgico. Especialmente, el término "predicción", como se usa aquí, se refiere a una evaluación individual de cualquier parámetro que pueda ser útil en determinar la evolución de un paciente. Como entenderán los expertos en la materia, la predicción de la respuesta clínica al tratamiento, aunque se prefiere que sea, no necesita ser correcta para el 100% de los sujetos a ser diagnosticados o evaluados. El término, sin embargo, requiere que se pueda identificar una parte estadísticamente significativa de los sujetos como que tienen una probabilidad aumentada de tener una respuesta positiva. El experto en la materia puede determinar fácilmente si un sujeto es estadísticamente significativo usando varias herramientas de evaluación estadística bien conocidas, por ejemplo, determinación de intervalos de confianza, determinación de los valores de p, prueba t de Student, prueba de Mann Whitney, etc. Los intervalos de confianza preferidos son al menos del 50%>, al menos del 60%>, al menos del 70%>, al menos del 80%>, al menos del 90%), al menos del 95%>. Los valores de p son, preferiblemente, 0,2, 0,1 ó 0,05. La predicción de la respuesta clínica se puede hacer utilizando cualquier criterio de valoración usado en oncología y conocido por el experto en la materia. "Prediction of the response" means, in the context of the present invention, the determination of the probability that the patient responds favorably or unfavorably to a particular therapy or treatment, including surgical treatment. Especially, the term "prediction", as used herein, refers to an individual evaluation of any parameter that may be useful in determining the evolution of a patient. As those skilled in the art will understand, the prediction of the clinical response to treatment, although it is preferred, does not need to be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant part of the subjects can be identified as having an increased probability of having a positive response. The person skilled in the art can easily determine if a subject is statistically significant using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann Whitney test, etc. . Preferred confidence intervals are at least 50%>, at least 60%>, at least 70%>, at least 80%>, at least 90%), at least 95%>. P values are preferably 0.2, 0.1 or 0.05. The prediction of the clinical response can be made using any assessment criteria used in oncology and known to the person skilled in the art.
A su vez, atendiendo al método de la presente invención, se podrían establecer otras subclasificaciones dentro de esta principal, facilitando, por tanto, la elección y el establecimiento de regímenes terapéuticos o tratamiento adecuados. Esta discriminación tal y como es entendida por un experto en la materia no pretende ser correcta en un 100% de las muestras analizadas. Sin embargo, requiere que una cantidad estadísticamente significativa de las muestras analizadas sean clasificadas correctamente. La cantidad que es estadísticamente significativa puede ser establecida por un experto en la materia mediante el uso de diferentes herramientas estadísticas, por ejemplo, pero sin limitarse, mediante la determinación de intervalos de confianza, determinación del valor significación P, test de Student o funciones discriminantes de Fisher, medidas no paramétricas de Mann Whitney, correlación de Spearman, regresión logística, regresión lineal, área bajo la curva de ROC (AUC). Preferiblemente, los intervalos de confianza son al menos del 90%, al menos del 95%, al menos del 97%, al menos del 98% o al menos del 99%. Preferiblemente, el valor de p es menor de 0,1 , de 0,05, de 0,01 , de 0,005 o de 0,0001 . Preferiblemente, la presente invención permite detectar correctamente la enfermedad de forma diferencial en al menos el 60%, más preferiblemente en al menos el 70%, mucho más preferiblemente en al menos el 80%, o aún mucho más preferiblemente en al menos el 90% de los sujetos de un determinado grupo o población analizada. El término "individuo", tal y como se utiliza en la descripción, se refiere a animales, preferiblemente mamíferos, y más preferiblemente, humanos. El término "individuo" no pretende ser limitativo en ningún aspecto, pudiendo ser éste de cualquier edad, sexo y condición física. In turn, according to the method of the present invention, other subclassifications could be established within this principal, thus facilitating the choice and establishment of appropriate therapeutic regimens or treatment. This discrimination, as understood by one skilled in the art, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly. The amount that is Statistically significant can be established by an expert in the field by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the significance value P, Student's test or Fisher's discriminant functions, measures non-parametric Mann Whitney, 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 value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention makes it possible to correctly detect the disease differentially by at least 60%, more preferably at least 70%, much more preferably at least 80%, or even much more preferably at least 90%. of the subjects of a certain group or population analyzed. The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" is not intended to be limiting in any aspect, and may be of any age, sex and physical condition.
La cantidad de referencia se obtiene a partir de los valores de expresión constitutiva de los genes, en un grupo de individuos sanos, o de la expresión de los genes en el grupo de individuos antes de ser sometidos al tratamiento. The reference amount is obtained from the constitutive expression values of the genes, in a group of healthy individuals, or from the expression of the genes in the group of individuals before being subjected to treatment.
La cantidad de referencia será, por ejemplo, en el caso de la diferenciación entre los pacientes afectados por cáncer de pulmón de los individuos sanos, la expresión constitutiva del gen o la cantidad de proteína detectada en un grupo control de individuos sanos. Sin embargo, en el caso de la subclasificación de los pacientes afectados por adenocarcinoma de pulmón o de los afectados por carcinoma de células escamosas, el grupo control estará formado por un grupo de enfermos con adenocarcinoma de pulmón que no tuvieron esa manifestación clínica. En otra realización preferida de este aspecto de la presente invención, la cantidad de referencia se obtiene a partir de una muestra de referencia. La cantidad de referencia puede obtenerse también, por ejemplo, de los límites de distribución normal de una cantidad encontrada en muestras obtenidas de una población de individuos con adenocarcinoma de pulmón, o carcinoma escamoso en distintas fases, mediante técnicas estadísticas bien conocidas. En otra realización preferida, la muestra de referencia se obtiene de los pacientes antes y después del tratamiento. En una realización preferida, el cáncer de pulmón es el cáncer de pulmón de células no pequeñas, y más preferiblemente es el carcinoma de células escamosas y adenocarcinomas. The reference amount will be, for example, in the case of differentiation between patients affected by lung cancer from healthy individuals, the constitutive expression of the gene or the amount of protein detected in a control group of healthy individuals. However, in the case of subclassification of patients affected by lung adenocarcinoma or those affected by squamous cell carcinoma, the control group will consist of a group of patients with lung adenocarcinoma who did not have that clinical manifestation. In another preferred embodiment of this aspect of the present invention, the reference amount is obtained from a reference sample. The reference quantity can also be obtained, for example, from the normal distribution limits of an amount found in samples obtained from a population of individuals with lung adenocarcinoma, or squamous carcinoma in different phases, using well-known statistical techniques. In another preferred embodiment, the reference sample is obtained from patients before and after treatment. In a preferred embodiment, lung cancer is non-small cell lung cancer, and more preferably it is squamous cell carcinoma and adenocarcinomas.
Otro aspecto de la invención se refiere a una composición farmacéutica que comprende un agente modulador de al menos uno de los genes que se seleccionan de entre PKP1, KRT15 y/o DSG3 para tratar un individuo diagnosticado de carcinoma escamoso según los métodos de la invención. Preferiblemente, el agente modulador es un inhibidor de la expresión de dichos genes. Another aspect of the invention relates to a pharmaceutical composition comprising a modulating agent of at least one of the genes that are selected from PKP1, KRT15 and / or DSG3 to treat an individual diagnosed with squamous carcinoma according to the methods of the invention. Preferably, the modulating agent is an inhibitor of the expression of said genes.
Otro aspecto de la invención se refiere a un anticuerpo para tratar un individuo diagnosticado de carcinoma escamoso, identificable por un método de la invención, donde el anticuerpo se selecciona de entre anti- PKP1 , anti- KRT15, y/o anti- DSG3. Another aspect of the invention relates to an antibody for treating an individual diagnosed with squamous carcinoma, identifiable by a method of the invention, wherein the antibody is selected from among anti-PKP1, anti-KRT15, and / or anti-DSG3.
Cuantificación de la proteína Protein quantification
La cuantificación de la cantidad de proteínas PKP1 , KRT15 y/o DSG3 puede hacerse por cualquiera de las técnicas conocidas por el experto en la materia, preferiblemente mediante técnicas inmmunológicas. The quantification of the amount of PKP1, KRT15 and / or DSG3 proteins can be done by any of the techniques known to those skilled in the art, preferably by immunological techniques.
En una realización más preferida, las técnicas inmunológicas están basadas en reacciones de precipitación, basadas en reacciones de aglutinación, inmunomarcación, radioinmunoanálisis y técnicas radioinmunométricas, ELISA {Enzime Linked ImmunoadSorbent Assay), o en cualquiera de sus combinaciones. En otra realización más preferida, las técnicas inmunológicas comprenden el inmunomarcaje. En otra realización aún más preferida, el inmunomarcaje se selecciona de entre inmunomarcaje con anticuerpos conjugados a enz ¡mas , inmunomarcaje con anticuerpos conjugados a fluorocromos, o citometría. Aún más preferiblemente, la citometría es citometría de flujo. KIT O DISPOSITIVO DE DIAGNÓSTICO, MICROARRAY o MICROARRAY DE PROTEÍNAS Y USOS In a more preferred embodiment, the immunological techniques are based on precipitation reactions, based on agglutination reactions, immunostaining, radioimmunoassay and radioimmunometric techniques, ELISA {Enzime Linked ImmunoadSorbent Assay), or any combination thereof. In another more preferred embodiment, immunological techniques comprise immunomarking. In another even more preferred embodiment, the immunomarking is selected from immunomarking with enzyme conjugated antibodies, immunomarking with fluorochrome conjugated antibodies, or cytometry. Even more preferably, the cytometry is flow cytometry. KIT OR DEVICE OF DIAGNOSIS, MICROARRAY or MICROARRAY OF PROTEINS AND USES
Otro aspecto de la presente invención se refiere a un kit o dispositivo, de aquí en adelante kit o dispositivo de la invención, que comprende los elementos necesarios para analizar el nivel de expresión de los genes PKP1, KRT15 y/o DSG3. En otra realización preferida el kit puede contener oligonucleótido diseñados a partir de una secuencia conocida o un ARNm de los genes, y/o capaces de hibridar con la secuencia de los genes PKP1, KRT15 y/o DSG3, para la posterior amplificación por PCR. Más preferiblemente, la secuencia de los genes PKP1 , KRT15 y/o DSG3 y es la secuencia nucleotídica SEO ID NO: 2, SEO ID NO: 4, y SEO ID NO: 6, respectivamente. Another aspect of the present invention relates to a kit or device, hereinafter kit or device of the invention, comprising the elements necessary to analyze the level of expression of the PKP1, KRT15 and / or DSG3 genes. In another preferred embodiment, the kit may contain oligonucleotides designed from a known sequence or an mRNA of the genes, and / or capable of hybridizing with the sequence of the PKP1, KRT15 and / or DSG3 genes, for subsequent PCR amplification. More preferably, the sequence of the PKP1, KRT15 and / or DSG3 genes is the nucleotide sequence SEO ID NO: 2, SEO ID NO: 4, and SEO ID NO: 6, respectively.
En otra realización preferida, el kit o dispositivo de la invención comprende al menos un anticuerpo anti- PKP1 , un anticuerpo anti- KRT15, y/o un anticuerpo anti- DSG3. En una realización preferida de este aspecto de la invención, el anticuerpo es humano, humanizado o sintético. En otra realización más preferida, el anticuerpo es monoclonal. En otra realización más preferida, el anticuerpo se encuentra marcado con un fluorocromo. Más preferiblemente el fluorocromo se selecciona de la lista que comprende Fluoresceína (FITC), Tetrametilrodamina y derivados, Ficoeritrina (PE), PerCP, Cy5, Texas, aloficocianina, o cualquiera de sus combinaciones. In another preferred embodiment, the kit or device of the invention comprises at least one anti-PKP1 antibody, an anti-KRT15 antibody, and / or an anti-DSG3 antibody. In a preferred embodiment of this aspect of the invention, the antibody is human, humanized or synthetic. In another more preferred embodiment, the antibody is monoclonal. In another more preferred embodiment, the antibody is labeled with a fluorochrome. More preferably, the fluorochrome is selected from the list comprising Fluorescein (FITC), Tetramethylrodamine and derivatives, Phycoerythrin (PE), PerCP, Cy5, Texas, allophycocyanin, or any combination thereof.
Más preferiblemente comprende los medios necesarios para comparar la cantidad detectada en el paso (b) con una cantidad de referencia Dicho kit puede contener todos aquellos reactivos necesarios para analizar el nivel de expresión de los genes PKP1, KRT15 y/o DSG3 por medio de cualquiera de los métodos descritos anteriormente en este documento. El kit además puede incluir, sin ningún tipo de limitación, tampones, agentes para prevenir la contaminación, inhibidores de la degradación de las proteínas, etc. Por otro lado el kit puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización. Preferiblemente, el kit comprende además las instrucciones para llevar a cabo cualquiera de los métodos de la invención. More preferably it comprises the means necessary to compare the amount detected in step (b) with a reference amount. Such kit may contain all those reagents necessary to analyze the expression level of the PKP1, KRT15 and / or DSG3 genes by means of any of the methods described earlier in this document. The kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit comprises furthermore the instructions for carrying out any of the methods of the invention.
En el caso de (a) un kit adecuado para la RQ-PCR, una técnica de cuantificación de la expresión génica sensible y reproducible, se desea que el kit comprenda adicionalmente un cebador del oligonucleótido poliT además del (de los) oligonucleótido(s) del kit. Estos reactivos pueden estar comprendidos opcionalmente en el kit. In the case of (a) a kit suitable for RQ-PCR, a technique for quantifying sensitive and reproducible gene expression, it is desired that the kit additionally comprises a polyT oligonucleotide primer in addition to the oligonucleotide (s) of the kit. These reagents may optionally be included in the kit.
Una Transferencia Northern implica el uso de electroforesis para separar las muestras de ARN por tamaño y la posterior detección con un(os) oligonucleótido(s) (sonda de hibridación) complementaria con (parte de) la secuencia diana del ARN de interés. A Northern Transfer involves the use of electrophoresis to separate RNA samples by size and subsequent detection with an oligonucleotide (s) (hybridization probe) complementary to (part of) the target sequence of the RNA of interest.
Es también posible que el(los) oligonucleótido(s) estén inmovilizados en manchas sobre una superficie (preferiblemente sólida). En una de sus realizaciones, el kit comprende una micromatriz, o micromatriz de la invención. Una micromatriz de ARN es una matriz sobre un sustrato sólido (normalmente un porta de vidrio o una celda de una película fina de silicio) que evalúa grandes cantidades de diferentes ARN que son detectables mediante sondas específicas inmovilizadas sobre manchas sobre un sustrato sólido. Cada mancha contiene una secuencia específica de ácido nucleico, normalmente una secuencia de ADN, como sondas (o indicadores). Aunque el número de manchas no está limitado de manera alguna, existe una realización preferida en la que la micromatriz se personaliza para los procedimientos de la invención. En una realización, dicha matriz personalizada comprende cincuenta manchas o menos, tal como treinta manchas o menos, incluyendo veinte manchas o menos. Por tanto, otro aspecto de la invención se refiere a una micromatriz que comprende oligonucleótidos diseñados a partir de una secuencia conocida o un ARNm de los genes, y/o capaces de hibridar con la secuencia de los genes PKP1, KRT15 y/o DSG3. Más preferiblemente, la secuencia de los genes PKP1, KRT15 y/o DSG3 es la secuencia nucleotídica SEQ ID NO: 2, SEO ID NO: 4, SEO ID NO: 6, respectivamente. It is also possible that the oligonucleotide (s) are immobilized in spots on a (preferably solid) surface. In one of its embodiments, the kit comprises a microarray, or microarray of the invention. An RNA microarray is a matrix on a solid substrate (usually a glass holder or a cell of a thin silicon film) that evaluates large amounts of different RNAs that are detectable by specific probes immobilized on spots on a solid substrate. Each spot contains a specific nucleic acid sequence, usually a DNA sequence, such as probes (or indicators). Although the number of spots is not limited in any way, there is a preferred embodiment in which the microarray is customized for the methods of the invention. In one embodiment, said custom matrix comprises fifty spots or less, such as thirty spots or less, including twenty spots or less. Therefore, another aspect of the invention relates to a microarray comprising oligonucleotides designed from a known sequence or an mRNA of the genes, and / or capable of hybridizing with the sequence of the PKP1, KRT15 and / or DSG3 genes. More preferably, the sequence of the PKP1, KRT15 and / or DSG3 genes is the nucleotide sequence SEQ ID NO: 2, SEO ID NO: 4, SEO ID NO: 6, respectively.
Otro aspecto de la invención se refiere a un microarray, de ahora en adelante microarray de la invención, que comprende oligonuleótidos o microarreglos de canal único diseñados a partir de una secuencia conocida o un ARNm de los genes PKP1, KRT15 y/o DSG3. Más preferiblemente, la secuencia de los genes PKP1, KRT15 y/o DSG3 es la secuencia nucleotídica SEO ID NO: 2, SEO ID NO: 4, SEO ID NO: 6, SEO ID NO: 8, SEO ID NO: 10, SEO ID NO: 12, SEO ID NO: 14, SEO ID NO: 16 y/o SEO ID NO: 18, respectivamente. Another aspect of the invention relates to a microarray, hereinafter microarray of the invention, comprising oligonuleotides or microarrays of single channel designed from a known sequence or an mRNA of the PKP1, KRT15 and / or DSG3 genes. More preferably, the sequence of the PKP1, KRT15 and / or DSG3 genes is the nucleotide sequence SEO ID NO: 2, SEO ID NO: 4, SEO ID NO: 6, SEO ID NO: 8, SEO ID NO: 10, SEO ID NO: 12, SEO ID NO: 14, SEO ID NO: 16 and / or SEO ID NO: 18, respectively.
Así, por ejemplo, las secuencias de oligonuleotidos son construidas en la superficie de un chip mediante el elongamiento secuencial de una cadena en crecimiento con un sólo nucleótido utilizando fotolitografía. Así, los oligonucleotidos son anclados por el extremo 3' mediante un método de activación selectiva de nucleotidos, protegidos por un reactivo fotolábil, mediante la incidencia selectiva de luz a través de una fotomáscara. La fotomáscara puede ser física o virtual. Thus, for example, oligonucleotide sequences are constructed on the surface of a chip by sequential elongation of a growing chain with a single nucleotide using photolithography. Thus, the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask. The photomask can be physical or virtual.
Así, las sondas oligonucleotidos pueden ser de entre 10 y 100 nucleotidos, más preferiblemente, de entre 20 y 70 nucleotidos, y aún más preferiblemente, de entre 24 y 30 nucleóitidos. Para la cuantificación de la expresión génica, preferiblemente se emplean aproximadamente unos 40 oligonucleotidos por gen. Thus, oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 30 nucleotides. For the quantification of gene expression, preferably about 40 oligonucleotides per gene are preferably used.
La síntesis in situ sobre un soporte sólido (por ejemplo, vidrio), podría hacerse mediante tecnología chorro de tinta (ink-jet), lo que requiere sondas más largas. Los soportes podrían ser, pero sin limitarse, filtros o membranas de NC o nylon (cargadas), silicio, o Portas de vidrio para microscopios cubiertos con aminosilanos, polilisina, aldehidos o epoxy. La sonda es cada una de las muestras del chip. El target es la muestra a analizar: RNA mensajero, RNA total, un fragmento de PCR, etc. Otro aspecto de la invención se refiere a un microarray de proteínas, de ahora en adelante microarray de proteínas de la invención, que comprende anticuerpos anti- PKP1 , anticuerpos anti- KRT15, y/o anticuerpos anti- DSG3. Las sondas son anticuerpos fijados a portaobjetos de vidrio y los blancos son muestras de suero o tejido. Otro aspecto de la invención se refiere al uso del kit o dispositivo, la micromatriz, o el microarray de la invención, de la para la obtención de datos útiles en el diagnóstico del adenocarcinoma de pulmón o del carcinoma escamoso. Synthesis in situ on a solid support (for example, glass) could be done using ink-jet technology, which requires longer probes. The supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy. The probe is each of the chip samples. The target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc. Another aspect of the invention relates to a protein microarray, hereinafter protein microarray of the invention, comprising anti-PKP1 antibodies, anti-KRT15 antibodies, and / or anti-DSG3 antibodies. The probes are antibodies fixed to glass slides and the targets are samples of serum or tissue. Another aspect of the invention relates to the use of the kit or device, the microarray, or the microarray of the invention, of data collection. useful in the diagnosis of lung adenocarcinoma or squamous carcinoma.
Otro aspecto de la invención se refiere a un medio de almacenamiento legible por un ordenador que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos de cualquiera de los métodos de la invención (del primer o del segundo método de la invención). Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of any of the methods of the invention (of the first or second method of the invention ).
Otro aspecto de la invención se refiere a una señal transmisible que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos de cualquiera de los métodos de la invención. Los términos "polinucleótido" y "ácido nucleico" se usan aquí de manera intercambiable, refiriéndose a formas poliméricas de nucleótidos de cualquier longitud, tanto ribonucleótidos (ARN ó RNA) como desoxiribonucleótidos (ADN ó DNA). Los términos "secuencia aminoacídica", "péptido", "oligopéptido", "polipéptido" y "proteína" se usan aquí de manera intercambiable, y se refieren a una forma polimérica de aminoácidos de cualquier longitud, que pueden ser codificantes o no codificantes, química o bioquímicamente modificados. Another aspect of the invention relates to a transmissible signal comprising program instructions capable of having a computer perform the steps of any of the methods of the invention. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA). The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, Chemically or biochemically modified.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived 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.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Fig. 1. Localización inmunohistoquímica de la proteína pKP1 en el carcinoma de pulmón de células escamosas o epidermoide. A) Tinción de la membrana. B) Tinción del citoplasma. C) Tinción del citoplasma con tinción intensa en el globo córneo (flecha). D) Intensa tinción nuclear en células con apariencia más inmadura. (A, B, E y F 40x; C 10x). Fig. 2. Localización inmunohistoquímica de la proteína pKP1 en el adenocarcinoma de pulmón. A) Tinción negativa B, C) Tinción en citoplasma. D) Tinción nuclear. (A y B, 40x; C y D 20x). Fig. 1. Immunohistochemical location of the pKP1 protein in squamous cell carcinoma or squamous cell carcinoma. A) Staining of the membrane. B) Cytoplasm staining. C) Cytoplasm staining with intense staining in the corneal balloon (arrow). D) Intense nuclear staining in cells with more immature appearance. (A, B, E and F 40x; C 10x). Fig. 2. Immunohistochemical localization of pKP1 protein in lung adenocarcinoma. A) Negative staining B, C) Staining in cytoplasm. D) Nuclear staining. (A and B, 40x; C and D 20x).
Fig. 3. Localización inmunohistoquímica de la proteína KRT15 en membranas del carcinoma de pulmón de células escamosas o epidermoides (40x). Fig. 3. Immunohistochemical location of the KRT15 protein in squamous cell lung carcinoma or epidermoid membranes (40x).
EJEMPLOS DE LA INVENCIÓN EXAMPLES OF THE INVENTION
A continuación se ilustrará la invención mediante unos ensayos realizados por los inventores, que pone de manifiesto la especificidad y efectividad de los métodos de la invención para obtener datos útiles en el diagnóstico diferencial del adenocarcinoma de pulmón y del carcinoma escamoso. The invention will now be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of the methods of the invention to obtain useful data in the differential diagnosis of lung adenocarcinoma and squamous carcinoma.
La localización inmunohistoquímica de las proteínas pKP1 , KRT15 y DSG3 se examinó en carcinoma de células escamosas y adenocarcinomas de pulmón. Setenta y cinco tumores de NSCLC se incluyeron en el estudio, que comprende 47 carcinomas escamosos y 28 adenocarcinomas. Los carcinomas de células escamosas fueron bien moderadamente diferenciado en 26 muestras y pobremente diferenciados en 21 muestras. Los adenocarcinomas fueron bien diferenciados moderadamente en 17 muestras y pobremente diferenciados en 1 1 muestras. The immunohistochemical location of the pKP1, KRT15 and DSG3 proteins was examined in squamous cell carcinoma and lung adenocarcinomas. Seventy-five NSCLC tumors were included in the study, comprising 47 squamous carcinomas and 28 adenocarcinomas. Squamous cell carcinomas were well moderately differentiated in 26 samples and poorly differentiated in 21 samples. The adenocarcinomas were well differentiated moderately in 17 samples and poorly differentiated in 1 1 samples.
Los resultados de inmunohistoquímica para la proteína pKP1 se resumen en la Tabla 1 . La localización inmunohistoquímica para pKP1 proteína se restringió principalmente a carcinomas escamosos, con una distribución heterogénea y la intensidad de la tinción entre los carcinomas diferentes y en diferentes áreas de un carcinoma. En carcinomas epidermoides la expresión de PKP 1 se localiza principalmente en el citoplasma y la membrana (Cohran, p = 0,001 ) dibujando las uniones intercelulares, (Fig. 1 ). La expresión membranosa de la proteína PKP1 se observó principalmente en los tumores bien-moderadamente diferenciados de carcinomas epidermoides (chi-cuadrado p = 0,176; Fisher p = 0,135), y considerando una neoplasia específica, la positividad se encontró predominantemente en las mejores zonas diferenciadas. La tinción nuclear de PKP1 fue observada en células con un aspecto más inmaduro (Fig. 1 D). The immunohistochemical results for the pKP1 protein are summarized in Table 1. The immunohistochemical localization for pKP1 protein was mainly restricted to squamous carcinomas, with a heterogeneous distribution and staining intensity between different carcinomas and in different areas of a carcinoma. In epidermoid carcinomas the expression of PKP 1 is mainly located in the cytoplasm and the membrane (Cohran, p = 0.001) drawing the intercellular junctions, (Fig. 1). Membranous expression of the PKP1 protein was mainly observed in well-moderately differentiated tumors of squamous cell carcinomas (chi-square p = 0.176; Fisher p = 0.135), and considering a specific neoplasm, positivity was found predominantly in the best differentiated areas. PKP1 nuclear staining was observed in cells with a more immature appearance (Fig. 1 D).
La tinción para PKP 1 en adenocarcinomas fue generalmente focal y se observó con una intensidad variable en el núcleo y en el citoplasma, y nunca en la membrana celular (Cohran, p = 0,039) (Fig. 2). Staining for PKP 1 in adenocarcinomas was generally focal and was observed with varying intensity in the nucleus and cytoplasm, and never in the cell membrane (Cohran, p = 0.039) (Fig. 2).
Los resultados de inmunohistoquímica para la proteína KRT15 se resumen en la Tabla 2. La localización inmunohistoquímica para la proteína KRT15 se restringió principalmente a carcinomas escamosos, con una distribución heterogénea y la intensidad de la tinción entre los carcinomas diferentes y en diferentes áreas de un carcinoma. La expresión de KRT15 se localizó en el citoplasma y en su mayoría en la membrana celular (Cohran, p = 0,000) dibujando las uniones intercelulares, (Fig. 3). En los carcinomas de células escamosas, tanto en los tumores bien- moderadamente como pobremente diferenciados (chi-cuadrado p = 1 ,000) mostraron tinción de la membrana celular para KRT15 y cuando se consideró una neoplasia específica, la positividad se observó principalmente en las mejores zonas diferenciadas. The immunohistochemical results for the KRT15 protein are summarized in Table 2. The immunohistochemical location for the KRT15 protein was mainly restricted to squamous carcinomas, with a heterogeneous distribution and staining intensity between different carcinomas and in different areas of a carcinoma. . The expression of KRT15 was located in the cytoplasm and mostly in the cell membrane (Cohran, p = 0.000) by drawing the intercellular junctions, (Fig. 3). In squamous cell carcinomas, both in well-moderately and poorly differentiated tumors (chi-square p = 1,000) showed staining of the cell membrane for KRT15 and when a specific neoplasm was considered, positivity was mainly observed in Better differentiated areas.
La tinción de la membrana celular para KRT15 fue generalmente ausente en los adenocarcinomas (Cohran, p = 0,005). La tinción positiva en este tipo de neoplasia fue generalmente focal y con una intensidad variable y se observaron en el núcleo y el citoplasma, pero nunca en la membrana celular. Se observó tinción nuclear para KRT15 en células con un aspecto más inmaduro. Staining of the cell membrane for KRT15 was generally absent in adenocarcinomas (Cohran, p = 0.005). Positive staining in this type of neoplasia was generally focal and with a variable intensity and was observed in the nucleus and cytoplasm, but never in the cell membrane. Nuclear staining for KRT15 was observed in cells with a more immature appearance.
Los resultados de inmunohistoquímica para la proteína DSG3 se resumen en la Tabla 3. La tinción inmunohistoquímica para proteína DSG3 fue principalmente ausente en adenocarcinomas, en cambio, carcinomas de células escamosas mostraron positividad en las membranas celulares (Cohran, p = 0,000). La expresión de DSG3 se observó principalmente en las membranas de carcinomas escamosos moderadamente bien diferenciados, (chi-cuadrado p = 0,792). Los adenocarcinomas mostraron ausencia de tinción para la proteína DSG3 ni en el núcleo, ni en el citoplasma ni en la membrana (Cohran, p = 0,223) y no hubo tinción en adenocarcinomas moderados ni pobremente diferenciados (Fisher, p = 1 ,000). The immunohistochemical results for the DSG3 protein are summarized in Table 3. Immunohistochemical staining for DSG3 protein was mainly absent in adenocarcinomas, however, squamous cell carcinomas showed positivity in cell membranes (Cohran, p = 0.000). DSG3 expression was mainly observed in the membranes of moderately well differentiated squamous carcinomas, (chi-square p = 0.792). The adenocarcinomas showed no staining for the DSG3 protein either in the nucleus, in the cytoplasm or in the membrane (Cohran, p = 0.223) and there was no staining in moderate or poorly differentiated adenocarcinomas (Fisher, p = 1,000).
Tabla 1 . Análisis de la localización de PKP1 .  Table 1 . PKP1 location analysis.
Tipo de tumor Núcleo PKP1 Citoplasma PKP1 Membrana PKP1 (N) + (N) - (N) + (N) - (N) + (N) - (N) Tumor type Core PKP1 Cytoplasm PKP1 Membrane PKP1 (N) + (N) - (N) + (N) - (N) + (N) - (N)
Adenocarcinoma 3.6% (1 ) 96.4% (27) 14.3% (4) 85.7% (24) 0% (0) 100% (28) (28) (Cochran p=0.039) (Cochran p=0.039) (Cochran p=0.039) Adenocarcinoma 3.6% (1) 96.4% (27) 14.3% (4) 85.7% (24) 0% (0) 100% (28) (28) (Cochran p = 0.039) (Cochran p = 0.039) (Cochran p = 0.039)
Carcinomas de 36.1 % (17) 63.8% (30) 59.6% (28) 40.4% (19) 57.4%(27) 42.5% (20) células escamosas Carcinomas of 36.1% (17) 63.8% (30) 59.6% (28) 40.4% (19) 57.4% (27) 42.5% (20) squamous cells
(Cochran p= 0.001 ) (Cochran p= 0.001 ) (Cochran p= 0.001 ) (Cochran p = 0.001) (Cochran p = 0.001) (Cochran p = 0.001)
(47) (47)
Carcinoma de Carcinoma of
células escamosas squamous cells
38.5% (10) 61 .5% (1 6) 65.4% (17) 34.6% (9) 69.2% (18) 30.8% (8) 38.5% (10) 61.5% (1 6) 65.4% (17) 34.6% (9) 69.2% (18) 30.8% (8)
Bien- Moderadamente Well- Moderately
diferenciados differentiated
(26) (26)
Carcinoma de 35% (7) 65% (13) 55% (1 1 ) 45% (9) 45% (9) 55% (1 1 ) células escamosas 35% carcinoma (7) 65% (13) 55% (1 1) 45% (9) 45% (9) 55% (1 1) squamous cells
Pobremente Poorly
(chi-square p= 1 .000) (chi-square p= 0.681 ) (chi-square p= 0.1 76; diferenciados  (chi-square p = 1, 000) (chi-square p = 0.681) (chi-square p = 0.1 76; differentiated
Fisher p= 0.135) Fisher p = 0.135)
(21 ) Tabla 1 . Análisis de la localización de PKP1 (cont.) (twenty-one ) Table 1 . PKP1 location analysis (cont.)
Figure imgf000026_0001
N= Número de casos
Figure imgf000026_0001
N = Number of cases
Tabla 2. Análisis de la localización de KRT15. Table 2. Analysis of the location of KRT15.
Figure imgf000027_0001
Figure imgf000027_0001
N= Número de casos Tabla 3. Análisis de la localización de DSG3. N = Number of cases Table 3. Analysis of the location of DSG3.
Figure imgf000028_0001
Figure imgf000028_0001
N= Número de casos N = Number of cases

Claims

REIVINDICACIONES
1 .- El uso de los genes PKP1, KRT15, DSG3, o de las proteínas PKP1 , KRT15, DSG3, para el diagnóstico diferencial del cáncer de pulmón. 1 .- The use of the PKP1, KRT15, DSG3, or PKP1, KRT15, DSG3 proteins, for the differential diagnosis of lung cancer.
2.- El uso de los genes PKP1, KRT15, DSG3, o de las proteínas PKP1 , KRT15, DSG3 según la reivindicación anterior, donde el cáncer de pulmón es cáncer de pulmón de células no pequeñas. 2. The use of the PKP1, KRT15, DSG3, or PKP1, KRT15, DSG3 proteins according to the preceding claim, wherein the lung cancer is non-small cell lung cancer.
3. - El uso de los genes PKP1, KRT15, DSG3, o de las proteínas PKP1 , KRT15, DSG3 según cualquiera de las reivindicaciones 1 -2, donde el cáncer de pulmón es el carcinoma de células escamosas. 3. - The use of the PKP1, KRT15, DSG3, or PKP1, KRT15, DSG3 proteins according to any of claims 1-2, wherein lung cancer is squamous cell carcinoma.
4. - Un método de obtención de datos útiles para el diagnóstico diferencial del cáncer de pulmón, que comprende: a. obtener una muestra biológica aislada que comprende células de un individuo, y b detectar simultáneamente el nivel de expresión de los genes PKP1,4. - A method of obtaining useful data for the differential diagnosis of lung cancer, comprising: a. obtain an isolated biological sample comprising cells of an individual, and b simultaneously detect the level of expression of PKP1 genes,
KRT15, DSG3, o la cantidad de las proteínas PKP1 , KRT15, DSG3, en la muestra aislada de (a), y c. comparar la expresión de los genes del paso (b) con una cantidad de referencia. KRT15, DSG3, or the amount of PKP1, KRT15, DSG3 proteins, in the sample isolated from (a), and c. compare the expression of the genes of step (b) with a reference amount.
5.- Un método para el diagnóstico diferencial del cáncer de pulmón, que comprende los pasos (a) - (c) según la reivindicación 4, y además comprende 5. A method for differential diagnosis of lung cancer, comprising steps (a) - (c) according to claim 4, and further comprising
(d) diagnosticar al individuo del paso (a) como un individuo con carcinomas escamoso, cuando presente una expresión aumentada del gen PKP1, KRT15 y/o DSG3 o una cantidad mayor de la proteína PKP1 , KRT15 y/o DSG3 en la muestra obtenida en (a), en relación a la cantidad de expresión detectada para dicho gen o dicha proteína en una población de pacientes de referencia. (d) diagnose the individual in step (a) as an individual with squamous carcinomas, when there is an increased expression of the PKP1, KRT15 and / or DSG3 gene or a larger amount of the PKP1, KRT15 and / or DSG3 protein in the sample obtained in (a), in relation to the amount of expression detected for said gene or said protein in a population of reference patients.
6. - El método según cualquiera de las reivindicaciones 4-5, donde la expresión aumentada del gen PKP1, o una cantidad mayor de la proteína PKP1 se detecta en el citoplasma y la membrana de las células. 6. - The method according to any of claims 4-5, wherein the increased expression of the PKP1 gene, or a larger amount of the PKP1 protein is detected in the cytoplasm and the cell membrane.
7. - El método según cualquiera de las reivindicaciones 4-6, donde el individuo del paso (a) se diagnostica como un individuo con adenocarcinoma de pulmón, cuando presenta la proteína PKP1 y/o KRT15 en el núcleo de la célula y está ausente en membrana. 7. - The method according to any of claims 4-6, wherein the individual of step (a) is diagnosed as an individual with lung adenocarcinoma, when he presents the PKP1 and / or KRT15 protein in the cell nucleus and is absent in membrane.
8. - El método según cualquiera de las reivindicaciones 4-7, donde el individuo del paso (a) se diagnostica como un individuo con adenocarcinoma, cuando no presenta la proteína DSG3 ni en la membrana, ni en el citoplasma, ni en el núcleo de la célula. 8. - The method according to any of claims 4-7, wherein the individual of step (a) is diagnosed as an individual with adenocarcinoma, when the DSG3 protein is not present in the membrane, in the cytoplasm, or in the nucleus of the cell.
9. - El método según cualquiera de las reivindicaciones 5-8, donde la muestra biológica aislada de un individuo del paso (a) es una muestra de tejido, preferiblemente tumoral. 9. - The method according to any of claims 5-8, wherein the biological sample isolated from an individual of step (a) is a tissue sample, preferably tumor.
10.- El método según cualquiera de las reivindicaciones 5-9, donde la detección de los niveles de expresión de los genes PKP1, KRT15 y/o DSG3 se realiza mediante Q-RT-PCR. 10. The method according to any of claims 5-9, wherein the detection of expression levels of the PKP1, KRT15 and / or DSG3 genes is performed by Q-RT-PCR.
1 1 . - El método según cualquiera de las reivindicaciones 5-10, donde la identificación de la cantidad de proteínas PKP1, KRT15 y/o DSG3, se realiza mediante técnicas inmmunológicas. eleven . - The method according to any of claims 5-10, wherein the identification of the amount of PKP1, KRT15 and / or DSG3 proteins, is performed by immunological techniques.
12. - El método según la reivindicación 1 1 , donde las técnicas inmunológicas están basadas en reacciones de precipitación, basadas en reacciones de aglutinación, inmunomarcación, radioinmunoanálisis y técnicas radioinmunométricas, ELISA {Enzime Linked ImmunoadSorbent Assay), o en cualquiera de sus combinaciones. 12. - The method according to claim 1, wherein the immunological techniques are based on precipitation reactions, based on agglutination reactions, immunostaining, radioimmunoassay and radioimmunometric techniques, ELISA {Enzime Linked ImmunoadSorbent Assay), or any combination thereof.
13. - El método según cualquiera de las reivindicaciones 1 1 -12, donde las técnicas inmunológicas comprenden el inmunomarcaje. 13. - The method according to any of claims 1 1 -12, wherein the immunological techniques comprise immunomarking.
14. - El método según la reivindicación 13, donde el inmunomarcaje se selecciona de entre inmunomarcaje con anticuerpos conjugados a enzimas , inmunomarcaje con anticuerpos conjugados a fluorocromos, o citometría. 14. - The method according to claim 13, wherein the immunomarking is selected from immunomarking with antibody conjugated to enzymes, immunomarking with antibodies conjugated to fluorochromes, or cytometry.
15. - El método según cualquiera de las reivindicaciones 4-14, donde el cáncer de pulmón es cáncer de pulmón de células no pequeñas. 15. - The method according to any of claims 4-14, wherein the lung cancer is non-small cell lung cancer.
16. - El método según cualquiera de las reivindicaciones 4-14, donde el cáncer de pulmón es el carcinoma de células escamosas. 16. - The method according to any of claims 4-14, wherein the lung cancer is squamous cell carcinoma.
17.- Un kit o dispositivo que comprende los elementos necesarios para analizar el nivel de expresión de los genes PKP1, KRT15 y/o DSG3 o la cantidad de proteína PKP1 , KRT15 y/o DSG3. 17.- A kit or device comprising the elements necessary to analyze the level of expression of the PKP1, KRT15 and / or DSG3 genes or the amount of PKP1, KRT15 and / or DSG3 protein.
18. - El kit o dispositivo según la reivindicación anterior, que comprende un un anticuerpo anti- PKP1 , un anticuerpo anti- KRT15, y/o un anticuerpo anti- DSG3. 18. - The kit or device according to the preceding claim, comprising an anti-PKP1 antibody, an anti-KRT15 antibody, and / or an anti-DSG3 antibody.
19. - El kit o dispositivo según cualquiera de las reivindicaciones 17-18, donde el anticuerpo es monoclonal 19. - The kit or device according to any of claims 17-18, wherein the antibody is monoclonal
20. - El kit o dispositivo según cualquiera de las reivindicaciones 17-19, donde el anticuerpo se encuentra marcado con un fluorocromo. 20. - The kit or device according to any of claims 17-19, wherein the antibody is labeled with a fluorochrome.
21 .- El kit o dispositivo según la reivindicación anterior, donde el fluorocromo se selecciona de la lista que comprende Fluoresceína (FITC), Tetrametilrodamina y derivados, Ficoeritrina (PE), PerCP, Cy5, Texas, aloficocianina, o cualquiera de sus combinaciones. 21. The kit or device according to the preceding claim, wherein the fluorochrome is selected from the list comprising Fluorescein (FITC), Tetramethylrodamine and derivatives, Phycoerythrin (PE), PerCP, Cy5, Texas, allophycocyanin, or any combination thereof.
22.- El uso del kit o dispositivo según cualquiera de las reivindicaciones 17-22, para el diagnóstico diferencial del cáncer de pulmón. 22. The use of the kit or device according to any of claims 17-22, for the differential diagnosis of lung cancer.
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