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WO2025051668A1 - Complexe arn-protéine en tant que nouveau biomarqueur pour le cancer du poumon - Google Patents

Complexe arn-protéine en tant que nouveau biomarqueur pour le cancer du poumon Download PDF

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Publication number
WO2025051668A1
WO2025051668A1 PCT/EP2024/074430 EP2024074430W WO2025051668A1 WO 2025051668 A1 WO2025051668 A1 WO 2025051668A1 EP 2024074430 W EP2024074430 W EP 2024074430W WO 2025051668 A1 WO2025051668 A1 WO 2025051668A1
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rpl10a
assay
lung cancer
rna
level
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Rastislav Horos
Tobias SIKOSEK
Jasmin SKOTTKE
Jessika CEILER
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Hummingbird Diagnostics GmbH
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Hummingbird Diagnostics GmbH
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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 relates to a method of diagnosing lung cancer in a patient.
  • the present invention further relates to a kit for carrying out this method.
  • Cancer is the uncontrolled growth of abnormal cells anywhere in a body.
  • the abnormal cells are termed cancer cells, malignant cells, or tumor cells. Cancer cells can proliferate uncontrollably and form a mass of cancer cells.
  • Lung cancer or bronchogenic carcinoma refers to tumors originating in the lung parenchyma or within the bronchi. It is one of the leading causes of cancer-related deaths in Europe and in the United States. Since 1987, lung cancer has been responsible for more deaths in women than breast cancer. It is estimated that there are 225,000 new cases of lung cancer in the United States annually, and approximately 160,000 die because of lung cancer. It is interesting to note that lung cancer was a relatively rare disease at the beginning of the 20th century. Its dramatic rise in later decades is attributable primarily to the increase in smoking among both males and females.
  • markers such as age, performance status and disease stage have been used to risk-stratify lung cancer patients and guide therapeutic decisions. These parameters provide some useful information, but more sensitive markers are clearly needed. Molecular and genetic studies have identified several such markers, which appear to play critical roles in carcinogenesis and affect patient outcomes. However, their prognostic and predictive value for lung cancer medicine has been low so far.
  • RNAs typically with a length of 18 to 50 nucleotides (nt) are a subgroup of short non-coding RNAs (18 to 200 nt). These RNAs can be found intracellularly and in extracellular environments, including body fluids such as blood.
  • a subclass of small non-coding RNAs, so called microRNAs (miRNAs) possess regulatory function exerted on the gene expression regulation of protein-encoding messenger RNAs (mRNAs).
  • miRNAs microRNAs
  • mRNAs protein-encoding messenger RNAs
  • RNA-binding proteins exert several RNA-related functions, e.g. RNA degradation, RNA transport, RNA storage, or RNA regulation.
  • rRNA 28S ribosomal RNA
  • RNP ribonucleoprotein
  • the present inventors have found that the 28S ribosomal RNA (rRNA) fragment having a nucleotide sequence according to SEQ ID NO: 1 is bound to the ribosomal protein L10A (RPL10A) and that this complex is detectable in the extracellular fraction of blood.
  • RPL10A ribosomal protein L10A
  • the present inventors could demonstrate that this complex is further found secreted by lung cancer cell lines and its prevalence is higher in medium conditioned by lung cancer cell lines as compared to cell-free fraction from healthy human individuals.
  • the present inventors could show that this complex is stable in different blood collection devices and under different storage conditions.
  • the detection of said complex will allow lung cancer diagnosis in patients. According to the present inventors, even the detection of the ribosomal protein L10A (RPL10A) alone appears to enable lung cancer diagnosis.
  • the present invention relates to a method of diagnosing lung cancer in a patient comprising the step of: determining the level of a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80% sequence identity thereto and a ribosomal protein L10A (RPL10A), and/or the level of ribosomal protein L10A (RPL10A) in an extracellular fraction of blood of a patient.
  • RNP ribonucleoprotein
  • the present invention relates to the use of a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80% sequence identity thereto and a ribosomal protein L10A (RPL10A), and/or a ribosomal protein L10A (RPL10A) for diagnosing lung cancer.
  • RNP ribonucleoprotein
  • the present invention relates to a kit for diagnosing lung cancer, said kit comprising: means for determining the level of a a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80% sequence identity thereto and a ribosomal protein L10A (RPL10A), and/or means for determining the level of ribosomal protein L10A (RPL10A) in an extracellular fraction of blood of a patient.
  • RNP ribonucleoprotein
  • the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kdlbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
  • the term “about” indicates a certain variation from the quantitative value it precedes.
  • the term “about” allows a ⁇ 5% variation from the quantitative value it precedes, unless otherwise indicated or inferred.
  • the use of the term “about” also includes the specific quantitative value itself, unless explicitly stated otherwise. For example, the expression “about 80°C” allows a variation of ⁇ 4°C, thus referring to range from 76°C to 84°C.
  • sequence alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res.
  • sequence matching may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX).
  • BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410.
  • BLAST protein searches are performed with the BLASTP program available e.g. on the web site: http://blast.ncbi.nlm.nih. go v/B last.
  • Preferred algorithm parameters used are the default parameters as they are set on the indicated web site:
  • compositional adjustments conditional compositional score matrix adjustment together with the database of non-redundant protein sequences (nr).
  • Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1: 154-162) or Markov random fields.
  • RNA molecule refers to a polymeric molecule essential in various biological roles in coding, decoding, regulation, and expression of genes.
  • RNA and deoxyribonucleic acid are nucleic acids.
  • DNA deoxyribonucleic acid
  • nucleic acids constitute one of the four major macromolecules essential for all known forms of life.
  • RNA is assembled as a chain of nucleotides, but unlike DNA, RNA is found in nature as a single strand folded onto itself, rather than a paired double strand.
  • RNA molecules play an active role within cells by catalysing biological reactions, controlling gene expression or sensing and communicating responses to cellular signals.
  • One of these active processes is protein synthesis, a universal function in which RNA molecules direct the synthesis of proteins on ribosomes. This process uses transfer RNA (tRNA) molecules to deliver amino acids to the ribosome, where ribosomal RNA (rRNA) then links amino acids together to form coded proteins.
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • Ribosomal ribonucleic acid refers to a type of noncoding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal DNA (rDNA) and then bound to ribosomal proteins to form small and large ribosome subunits. rRNA is the physical and mechanical factor of the ribosome that forces transfer RNA (tRNA) and messenger RNA (mRNA) to process and translate the latter into proteins. Ribosomal RNA is the predominant form of RNA found in most cells; it makes up about 80% of cellular RNA despite never being translated into proteins itself. Ribosomes are composed of approximately 60% rRNA and 40% ribosomal proteins by mass.
  • Mammalian cells have 2 mitochondrial (12S and 16S) rRNA molecules and 4 types of cytoplasmic rRNA molecules (the 28S, 5.8S, 18S, and 5S subunits).
  • the 28S, 5.8S, and 18S rRNAs are encoded by a single transcription unit (45S) separated by 2 internally transcribed spacers.
  • the first spacer corresponds to the one found in bacteria and archaea, and the other spacer is an insertion into what was the 23S rRNA in prokaryotes.
  • the 45S rDNA is organized into 5 clusters (each has 30-40 repeats) on chromosomes 13, 14, 15, 21, and 22. These are transcribed by RNA polymerase I.
  • the DNA for the 5S subunit occurs in tandem arrays (-200-300 true 5S genes and many dispersed pseudogenes), the largest one on the chromosome lq41-42.
  • 5S rRNA is transcribed by RNA polymerase III.
  • the 18S rRNA in most eukaryotes is in the small ribosomal subunit, and the large subunit contains three rRNA species (the 5S, 5.8S, and 28S).
  • rRNA 28S ribosomal RNA
  • RNP ribonucleoprotein
  • 28S ribosomal RNA refers to a structural ribosomal RNA (rRNA) for the large subunit (LSU) of eukaryotic cytoplasmic ribosomes, and thus, to one of the basic components of all eukaryotic cells. It has a size of 28S in mammals, hence the name.
  • ribonucleoprotein (RNP) complex refers to a protein complex containing a ribonucleic acid (RNA).
  • RNP complex is found in all living organisms.
  • a ribonucleoprotein complex includes, for example, the subunits of ribosomes, the spliceosome, telomerase, snRNPs and hnRNPs, the nuclease RNase P, and various transcription initiation factors, all of which are localized in the nucleus.
  • methylated RNA refers to RNA which has been post- transcriptionally edited or modified by methylation.
  • the methylation involves the transfer of a methyl group from an active methyl compound to another compound. More than 160 modifications have been found in mRNA and non-coding RNA species.
  • Reported roles of RNA methylation include RNA splicing stability, nuclear export, translation, initiation of miRNA biogenesis, immunogenicity, DNA damage repair, perturbation of cellular differentiation, sex determination, spermatogensesis, embryonic development, learning, memory, immune response, and several human and animal diseases.
  • the methylation can occur at a base (e.g.
  • RNA occurring at a base is preferably selected from the group consisting of 6-methyladenosine (m 6 A), 5 -methylcytidine (m 5 C), 5-methyluridine (m 5 U), 3-methyluridine (m 3 U), 1 -methyladenosine (m ⁇ A), and 1 -methylguanosine (m ⁇ G), or is a combination thereof.
  • the 2'-O-methylation of the backbone ribose is the most common and conserved type of small non-coding RNA modification.
  • 2'-O-methylation refers to a common nucleoside modification of RNA, where a methyl group is added to the 2’hydroxyl of the ribose moiety of a nucleoside, producing a methoxy group.
  • 2'-O-methylated nucleosides are mostly found in ribosomal RNA and small nuclear RNA and occur in the functionally essential regions of the ribosome and spliceosome.
  • the present inventors have found that the 28S ribosomal RNA (rRNA) fragment/molecule having a nucleotide sequence according to SEQ ID NO: 1 ( CGCCGGU GA A AU ACC ACU) is bound to the ribosomal protein L10A (RPL10A) and that this complex is detectable in the extracellular fraction of blood.
  • rRNA 28S ribosomal RNA
  • RPL10A ribosomal protein L10A
  • the detection of said complex will allow lung cancer diagnosis in patients. According to the present inventors, even the detection of the ribosomal protein L10A (RPL10A) alone appears to enable lung cancer diagnosis.
  • RPL10A ribosomal protein L10A
  • ribosomal protein L10A refers to a protein that is encoded by the RPL10A gene.
  • the RPL10A gene encodes a 217 amino acid protein.
  • the protein has a molecular weight of about 25 Da.
  • Ribosomes the organelles that catalyse protein synthesis, consists of a small 40S subunit and a large 60S subunit.
  • the ribosomal protein L10A (RPL10A) is a component of the 60S subunit. It belongs to the LIP family of ribosomal proteins. Specifically, RPL10A has one domain called the ribosomal LI superfamily domain. There are 13 conserved mRNA/rRNA interface residues on this domain.
  • RPL10A is required for joining the 60S and 40S ribosomal subunits into the functional 80S ribosome.
  • RPL10A is common and detected across all eukaryotic groups. The number and sequence of amino acids are highly conserved among animals, fungi, plants, and protists.
  • RPL10A is found on the exit tunnel of 80S ribosomes and preferentially provides the translation of specific subsets of mRNAs by binding to Internal Ribosome Entry Sites (IRES) on the mRNAs.
  • the ribosomal protein L10A is human ribosomal protein L10A (RPL10A).
  • ribosomal proteins In the latest ribosomal proteins nomenclature, it is also denoted under the name “large ribosomal subunit protein uLl” protein.
  • the human uLl protein has the Accession number NP_009035 (Version NP_009035.3).
  • variants/precursors of the nucleotide sequence according to SEQ ID NO: 1 ( CGCCGGU GA A AU ACC ACU) (“core sequence”) allow lung cancer diagnosis.
  • the variants/precursors of the nucleotide sequence according to SEQ ID NO: 1 ( CGCCGGU GA A AU ACC ACU) as described herein have the following nucleotide sequences:
  • RNA molecule comprising the nucleotide sequence according to SEQ ID NO: 1 ( CGCCGGU GA A AU ACC ACU) (“core sequence”) also the nucleotide sequences according to SEQ ID NO: 2 to SEQ ID NO: 5 are covered.
  • sequences according to SEQ ID NO: 1 to SEQ ID NO: 5 are preferably human sequences.
  • nucleotide sequences can be present in a 2’ -ortho-methylated or 2’-ortho- non-methylated state and optionally possess various 5’ ends, such as 5’ phosphorylation or 5’ hydroxyl and/or various 3’ ends, such as 3’ hydroxyl, 3’ phosphate or 3’, 2’ cyclic phosphate.
  • cancer refers to or describes a physiological condition in an individual that is typically characterized by unregulated cell growth.
  • cancer also encompasses cancer metastases.
  • lung cancer refers to a disease which consists of uncontrolled cell growth in tissues of the lung. This growth may lead to metastasis, which is the invasion of adjacent tissue and infiltration beyond the lungs.
  • metastasis which is the invasion of adjacent tissue and infiltration beyond the lungs.
  • the vast majority of primary lung cancers are carcinomas of the lung, derived from epithelial cells. Lung cancer is the most common cause of cancer-related death in men and women. The most common symptoms are shortness of breath, coughing (including coughing up blood), and weight loss.
  • the lung cancer e.g. lung cancer of state I, II, III, or IV
  • NSCLC non-small-cell lung carcinoma
  • SCLS small-cell lung carcinoma
  • Lung cancer can be staged as follows:
  • Stage 1 means that the cancer is small. It hasn’t spread to the lymph nodes or other distant organs.
  • Stage I belongs to the early lung cancer stages. Stage I can be divided into IA and IB. Stage IA means the cancer is 3 cm or smaller.
  • Stage IB means the cancer is between 3 cm and 4 cm. It might also be growing into structures such as: the main airway of the lung (main bronchus) or the membrane covering the lung (visceral pleura).
  • Stage 11 still belongs to the early lung cancer stages. Stage II can be divided into stage IIA and IIB. Part of the affected lung might have collapsed.
  • Stage IIA means that the cancer is between 4 cm and 5 cm in size but there are no cancer cells in any lymph nodes.
  • Stage IIB means that the cancer is up to 5 cm in size and there are cancer cells in the lymph nodes close to the affected lung. Alternatively, it is between 5 cm and 7 cm but there are no cancer cells in any lymph nodes.
  • the cancer is not in any lymph nodes but has spread into one or more of the following areas: the chest wall (ribs, muscle or skin), the nerve close to the lung (the phrenic nerve), or the layers that cover the heart (mediastinal pleura and parietal pericardium).
  • the cancer is less than 7 cm but there is more than one tumor in the same lobe of the lung.
  • Stage III can be divided into stage IIIA, IIIB and IIIC. It is sometimes called locally advanced lung cancer.
  • the cancer is up to 5cm in size and has spread to the lymph nodes in the center of the chest on the same side as the tumor.
  • the cancer it is between 5 cm and 7 cm and there is more than one tumor in the same lobe of the lung.
  • the cancer has spread into one or more of the following areas just outside the lung: the chest wall (ribs, muscle or skin), the nerve close to the lung (the phrenic nerve), the layers that cover the heart (mediastinal pleura and parietal pericardium), or lymph nodes in the lung or close to the lung.
  • the cancer is larger than 7 cm.
  • lymph nodes It hasn't spread into lymph nodes but has spread into one or more of the following areas: the muscle under the lung (diaphragm), the center area of the chest (mediastinum), the heart, a main blood vessel, the wind pipe (trachea), the nerve that goes to the voice box (larynx), the food pipe (oesophagus), a spinal bone, or the area where the wind pipe divides (the carina).
  • the cancer is in more than one lobe of the same lung and there might also be cancer cells in lymph nodes close to the affected lung.
  • Stage IIIB can also mean different things.
  • the cancer is less than 5 cm and has spread into lymph nodes in one of these places: the opposite side of the chest from the affected lung, the neck, or above the collarbone.
  • the cancer is between 5 cm to 7 cm and has spread into lymph nodes in the center of the chest.
  • the cancer is any size, has spread into lymph nodes in the center of the chest, and has spread into one or more of the following areas: the chest wall, the muscle under the lung (diaphragm), or the layers that cover the heart (mediastinal pleura and parietal pericardium).
  • the cancer has spread into the lymph nodes in the center of the chest.
  • the lung tumor is more than 7 cm or it has spread into a major structure in your chest such as: the heart, the wind pipe (trachea), the food pipe (oesophagus), or a main blood vessel.
  • Stage IIIC means the cancer is between 5 cm and 7 cm in size or has spread into one or more of the following: the nerve close to the lung (phrenic nerve) or the covering of the heart (parietal pericardium) and it has spread into lymph nodes: in the center of the chest on the opposite side from the affected lung or at the top of the lung on the same side or opposite side or above the collar bone.
  • the nerve close to the lung phrenic nerve
  • the covering of the heart parietal pericardium
  • lymph nodes in the center of the chest on the opposite side from the affected lung or at the top of the lung on the same side or opposite side or above the collar bone.
  • lymph nodes in the center of the chest on the opposite side from the affected lung or at the top of the lung on the same side or opposite side or above the collar bone.
  • there is more than one tumor in a different lobe of the same lung there is more than one tumor in a different lobe of the same lung.
  • stage IIIC can mean the cancer is bigger than 7 cm or it has spread into one of the following: the muscle under the lung (the diaphragm), the center of the chest (mediastinum), the heart, a major blood vessel, the wind pipe (trachea), the nerve going to the voice box (the recurrent laryngeal nerve), the food pipe (oesophagus), a spinal bone, or the area where the windpipe divides (the carina) and it has spread into lymph nodes: in the center of the chest on the opposite side from the affected lung or at the top of the lung on the same side or opposite side or above the collar bone.
  • Stage IV means that the lung cancer has spread. It can be designated as advanced lung cancer. It is divided into stage IVA and IVB.
  • Stage IVA can mean any of the following: there is cancer in both lungs, the cancer is in the covering of the lung (the pleura) or the covering of the heart (pericardium), or there is fluid around the lungs or the heart that contains cancer cells. Alternatively, it can mean that there is a single area of cancer that has spread outside the chest to a lymph node or to an organ such as the liver or bone.
  • Stage IVB means that the cancer has spread to several areas in one or more organs.
  • early stage lung cancer refers to lung cancer that is early in its growth. It has not grown deeply into nearby tissues. In addition, it has not spread to other parts of the body of a patient. No metastasis has been formed. Specifically, early stage lung cancer is lung cancer of stage I.
  • small-cell lung carcinoma refers to a type of highly malignant cancer that arises within the lungs.
  • Small-cell carcinoma of the lungs usually presents in the central airways and infiltrates the submucosa leading to narrowing of bronchial airways.
  • Common symptoms include cough, dyspnea, weight loss, and debility.
  • small cell carcinoma has a shorter doubling time, higher growth fraction, and earlier development of metastases.
  • SCLC accounts for about 15% of all lung cancers.
  • Small-cell lung carcinoma can be divided into two clinicopathological stages, termed limited stage (LS) and extensive stage (ES).
  • the stage is generally determined by the presence or absence of metastases, whether or not the tumor appears limited to the thorax, and whether or not the entire tumor burden within the chest can feasibly be encompassed within a single radiotherapy portal.
  • the cancer is said to be LS. If cancer has spread beyond that, it is said to be ES.
  • non-small-cell lung carcinoma refers to any type of epithelial lung cancer other than small-cell lung carcinoma (SCLC).
  • SCLC small-cell lung carcinoma
  • NSCLC accounts for about 85% of all lung cancers.
  • NSCLCs are relatively insensitive to chemotherapy, compared to small-cell lung carcinomas. When possible, they are primarily treated by surgical resection with curative intent, although chemotherapy has been used increasingly both preoperatively (neoadjuvant chemotherapy) and postoperatively (adjuvant chemotherapy).
  • NSCLC is squamous-cell carcinoma, large-cell carcinoma, or adenocarcinoma.
  • diagnosing lung cancer means determining whether a patient shows signs of or suffers from lung cancer.
  • (therapeutic) treatment/therapy relates to any treatment/therapy which improves the health status and/or prolongs (increases) the lifespan of a patient.
  • Said treatment/therapy may eliminate the disease in a patient, arrest, inhibit, or slow the development of a disease in a patient, decrease the frequency or severity of symptoms in a patient, and/or decrease the recurrence in a patient who currently has or who previously has had a disease.
  • lung cancer therapy refers to any protocol, method, and/or agent that can be used in the prevention, management, treatment, and/or amelioration of lung cancer.
  • lung cancer therapy means accomplishing one or more of the following: (i) tumor growth inhibition and/or tumor cell death, (ii) reduction of tumor marker(s), (iii) reduction of tumor lesions and metastases, (iv) reduction of tumor burden as evidenced by imaging studies (e.g. computer tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), etc.), and (v) reduction of tumor burden as evidenced by clinical appraisal or self-report by the patient.
  • CT computer tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • patient refers to any subject for whom it is desired to know whether she or he suffers from lung cancer.
  • patient refers to a subject suspected to be affected by lung cancer.
  • the patient may be diagnosed to be affected by lung cancer, i.e. diseased, or may be diagnosed to be not affected by lung cancer, i.e. healthy.
  • the patient may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human individuals are particularly preferred.
  • control subject refers to a subject known to be not affected by lung cancer (negative control), i.e. healthy.
  • control subject also refers to a subject known to be affected by lung cancer (positive control), i.e. diseased.
  • control subject which is known to be healthy, i.e. not suffering from lung cancer, may possibly suffer from another disease or condition not tested/known.
  • the (control) subject may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human healthy individuals are particularly preferred.
  • whole blood refers to a blood product which contains all physiologically occurring blood components, i.e. cells, proteins, and other components.
  • Whole blood is blood from a standard blood donation. Specifically, whole blood is made up of erythrocytes, leukocytes, and thrombocytes, all of which are suspended in a liquid called plasma. More specifically, whole blood encompasses the cellular (erythrocytes, leukocytes, and thrombocytes) components as well as the extracellular (serum and plasma) components of blood.
  • a whole blood sample may be provided by removing blood from a patient or (control) subject, but may also be provided by using a previously isolated sample. For example, a blood sample may be taken from a patient or (control) subject by conventional blood collection techniques.
  • the blood sample is obtained from at least one (control) subject, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or 1,000 (control) subject(s), it is designated as “reference blood sample”.
  • the reference blood sample is from the same source than the blood sample of the patient to be tested.
  • the blood sample from the patient to be tested is an extracellular fraction of blood and the (reference) blood sample from the control subject is also an extracellular fraction of blood. It is further preferred that both are from the same species, e.g. from a human.
  • the measurements of the reference blood sample of the (control) subject and the blood sample of the patient to be tested are identical, e.g. both have an identical volume. It is particularly preferred that the reference blood sample and the blood sample are from (control) subjects/patients of the same sex and similar age.
  • the whole blood sample may be collected by means of a blood collection tube. It is, for example, collected in a PAXgene Blood RNA tube, in a Tempus Blood RNA tube, in an EDTA-tube, Serum- Monovette tube, in a Na-citrate tube, Heparin-tube, or in an ACD-tube (Acid citrate dextrose).
  • the whole blood sample may be collected in a blood collection tube containing cell- free nucleic acid stabilizing chemical agents, such as glutaraldehyde, formaldehyde, or similar (e.g.
  • the whole blood sample may also be collected by means of a bloodspot technique, e.g. using a Mitra Microsampling Device. This technique requires smaller sample volumes, typically 45-60 pl for humans or less.
  • the whole blood may be extracted from the patient via a finger prick with a needle or lancet.
  • the whole blood sample may have the form of a blood drop. Said blood drop is then placed on an absorbent probe, e.g.
  • RNA molecule as described herein, are extracted from the dried blood spots into a supernatant which is then further analyzed. In this way, the level of the RNA molecule is determined. This technique is suitable for monitoring patients having lung cancer at home (on a home care/home sampling basis) or for screening purposes.
  • extracellular fraction of blood refers to the acellular portion of blood which is produced from whole blood by removing the cellular fraction (erythrocytes, leukocytes, and thrombocytes). In other words, the acellular portion of blood is depleted of the cellular blood components (erythrocytes, leukocytes, and thrombocytes).
  • the cellular fraction of blood comprises/essentially consists of/consists of erythrocytes, leukocytes, and thrombocytes.
  • the extracellular fraction of blood structurally differs from whole blood.
  • the extracellular fraction of blood is only composed of the acellular (serum and plasma) components of blood.
  • the extracellular fraction of blood is free of the cellular (erythrocytes, leukocytes, and thrombocytes) components of blood.
  • serum and plasma represent the extracellular components of blood.
  • the extracellular fraction of blood is often denoted as “cell- free blood”.
  • cellular fraction of blood refers to the cellular portion of blood which is produced from whole blood by removing the extracellular fraction (serum and plasma).
  • the cellular fraction of blood is depleted of the extracellular blood components (serum and plasma).
  • the cellular portion of blood comprises/essentially consists of/consists of erythrocytes, leukocytes, and thrombocytes.
  • blood plasma refers to the liquid component of blood in which the blood cells are absent, but which contains proteins and other constituents of whole blood in suspension. It makes up about 55% of the body's total blood volume. It is the intravascular part of extracellular fluid (all body fluid outside cells). It is mostly water (up to 95% by volume), and contains important dissolved proteins (6-8%, e.g. serum albumins, globulins, and fibrinogen), glucose, clothing factors, electrolytes (Na + , Ca 2+ , Mg 2+ , HCO3 , CF, etc.), hormones, carbon dioxide (plasma being the main medium for excretory product transportation), and oxygen.
  • proteins e.g. serum albumins, globulins, and fibrinogen
  • glucose e.g. serum albumins, globulins, and fibrinogen
  • clothing factors e.g. serum albumins, globulins, and fibrinogen
  • electrolytes Na + , Ca 2+ , Mg 2+
  • Blood plasma is obtained from whole blood by removing the blood cells (erythrocytes, leukocytes, and thrombocytes) by centrifugation in the presence of an anticoagulant. During the centrifugation process, the blood cells fall to the bottom of the tube. The blood plasma (supernatant) is then poured or drawn off.
  • blood cells erythrocytes, leukocytes, and thrombocytes
  • blood serum refers to the liquid component of blood which does not play a role in clotting. It may be defined as blood plasma without the clotting factors, or as blood with all cells and clotting factors removed. Serum includes all proteins not used in blood clotting, all electrolytes, antibodies, antigens, hormones and potential exogenous substances. Serum does not contain erythrocytes, leukocytes, thrombocytes, and clotting factors anymore. To obtain blood serum, a blood sample is allowed to clot (coagulation). The sample is then centrifuged to remove the clot and blood cells, and the resulting liquid supernatant is serum.
  • the level of the ribonucleoprotein (RNP) complex and/or the level of RPL10A is determined in an extracellular fraction of blood of a patient.
  • level refers to an amount (measured for example in grams, mole, or ion counts) or concentration (e.g. absolute or relative concentration) of the RNP complex.
  • concentration e.g. absolute or relative concentration
  • level also comprises scaled, normalized, or scaled and normalized amounts or values (e.g. units/ml or mol/ml).
  • the determined ribonucleoprotein (RNP) complex level is compared with an appropriate ribonucleoprotein (RNP) complex “reference level”. Specifically, the level of the ribonucleoprotein (RNP) complex is compared to a reference level of said ribonucleoprotein (RNP) complex. More specifically, the reference level of the ribonucleoprotein (RNP) complex is determined in an extracellular fraction of blood of (control) subjects.
  • the reference level may also be a cut-off or threshold level.
  • a cut-off or threshold level can be determined experimentally, empirically, or theoretically.
  • a cut-off or threshold level can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art.
  • the cut-off or threshold level must be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity (and so the threshold level) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • ROC curve receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biomarker tests.
  • the ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cutoff or threshold levels are set as continuous variables to calculate a series of sensitivity and specificity values.
  • sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC > 0.5, the predicted/prognosed/diagnosed result gets better and better as AUC approaches 1.
  • the level of the RNP complex is preferably determined by an immunoprecipitation-based assay or by an RNA-pulldown assay.
  • the immunoprecipitation-based assay is an antibody-based pull-down of RPL10A followed by the detection of the co-immunoprecipitated RNA molecule.
  • the detection of the co-immunoprecipitated RNA molecule is carried out by a Polymerase Chain Reaction (PCR) or next generation sequencing (NGS).
  • the PCR is selected from the group consisting of digital PCR, real-time PCR (quantitative PCR or qPCR), preferably TaqMan qPCR, multiplex PCR, nested PCR, high fidelity PR, fast PCR, hot start PCR, and GC-rich PCR.
  • the RNA-pulldown assay is an antisense oligonucleotide-based pull-down of the RNA molecule followed by the detection of RPL10A.
  • the detection of RPL10A is carried out by an antibody-based assay or a mass spectrometry-based assay.
  • the antibody-based assay is selected from the group consisting of an Enzyme- Linked Immunosorbent Assay (ELISA), an Immuno-Polymerase Chain Reaction (Immuno-PCR) assay, an Immuno-Next generation sequencing (Immuno-NGS) assay, and a Western Blot assay or the mass spectrometry-based assay is selected from the group consisting of a liquid chromatography-mass spectrometry (LC-MS) assay and fractionation followed by mass spectrometry (F/MS).
  • ELISA Enzyme- Linked Immunosorbent Assay
  • Immuno-PCR Immuno-Polymerase Chain Reaction
  • Immuno-NGS Immuno-Next generation sequencing
  • Western Blot assay or the mass spectrometry-based assay is selected from the group consisting of a liquid chromatography-mass spectrometry (LC-MS) assay and fractionation followed by mass spectrometry (F/MS).
  • PCR polymerase chain reaction
  • the PCR is preferably selected from the group consisting of digital PCR, real-time PCR (quantitative PCR or qPCR), preferably TaqMan qPCR, multiplex PCR, nested PCR, high fidelity PR, fast PCR, hot start PCR, and GC-rich PCR.
  • NGS next generation sequencing
  • SGS second generation sequencing
  • MPS massively parallel sequencing
  • POCT point-of-care testing
  • POCT is often accomplished through the use of transportable, portable, and handheld instruments and test kits.
  • Small bench analyzers or fixed equipment can also be used when a handheld device is not available - the goal is to collect the specimen and obtain the results in a very short period of time at or near the location of the individual so that the treatment plan can be adjusted as necessary before the individual leaves the hospital.
  • kit of parts in short: kit
  • kit is understood to be any combination of at least some of the components identified herein, which are combined, coexisting spatially, to a functional unit, and which can contain further components.
  • Lung cancer or bronchogenic carcinoma refers to tumors originating in the lung parenchyma or within the bronchi. It is one of the leading causes of cancer-related deaths in Europe and in the United States. Since 1987, lung cancer has been responsible for more deaths in women than breast cancer. It is estimated that there are 225,000 new cases of lung cancer in the United States annually, and approximately 160,000 die because of lung cancer. It is interesting to note that lung cancer was a relatively rare disease at the beginning of the 20th century. Its dramatic rise in later decades is attributable primarily to the increase in smoking among both males and females. Historically, markers such as age, performance status and disease stage have been used to risk-stratify lung cancer patients and guide therapeutic decisions. These parameters provide some useful information, but more sensitive markers are clearly needed. Molecular and genetic studies have identified several such markers, which appear to play critical roles in carcinogenesis and affect patient outcomes. However, their prognostic and predictive value for lung cancer medicine has been low so far.
  • RNAs typically with a length of 18 to 50 nucleotides (nt) are a subgroup of short non-coding RNAs (18 to 200 nt). These RNAs can be found intracellularly and in extracellular environments, including body fluids such as blood.
  • a subclass of small non-coding RNAs, so called microRNAs (miRNAs) possess regulatory function exerted on the gene expression regulation of protein-encoding messenger RNAs (mRNAs).
  • miRNAs microRNAs
  • mRNAs messenger RNAs
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • snoRNA small nucleolar RNA
  • a plethora of short fragments with no described regulatory function can be readily found in biological RNA samples. This is likely result of (active or passive) fragmentation of longer RNA precursors.
  • RNA molecules may exist in cells in the form of ribonucleoprotein (RNP) complexes.
  • RNP ribonucleoprotein
  • RNA-binding proteins exert several RNA-related functions, e.g. RNA degradation, RNA transport, RNA storage, or RNA regulation.
  • rRNA 28S ribosomal RNA
  • RNP ribonucleoprotein
  • the present inventors have found that the 28S ribosomal RNA (rRNA) fragment having a nucleotide sequence according to SEQ ID NO: 1 is bound to the ribosomal protein L10A (RPL10A) and that this complex is detectable in the extracellular fraction of blood.
  • the present inventors could demonstrate that this complex is further found secreted by lung cancer cell lines and its prevalence is higher in medium conditioned by lung cancer cell lines as compared to cell- free fraction from healthy human individuals.
  • the present inventors could show that this complex is stable in different blood collection devices and under different storage conditions.
  • the detection of said complex will allow lung cancer diagnosis in patients. According to the present inventors, even the detection of the ribosomal protein L10A (RPL10A) alone appears to enable lung cancer diagnosis.
  • the present invention relates to a (an) (in vitro) method of diagnosing lung cancer in a patient comprising the step of: determining the level of a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises an RNA molecule comprising/having a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g.
  • RNP ribonucleoprotein
  • RPL10A ribosomal protein L10A
  • RPL10A ribosomal protein L10A
  • RNA molecule comprising
  • nucleotide sequence that is a fragment of the nucleotide sequence according to (i), preferably a nucleotide sequence that is a fragment which is between 1 to 6, more preferably between 1 to 4, and most preferably between 1 to 2, e.g. 1, 2, 3, 4, 5, or 6, nucleotide(s) shorter than the nucleotide sequence according to (i), or
  • nucleotide sequence that has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the nucleotide sequence according to (i) or nucleotide sequence fragment according to (ii) is part of the RNP complex whose level is determined in the extracellular fraction of blood of a patient.
  • the level of the RNP complex is compared to a reference level of said RNP complex, and/or the level of RPL10A is compared to a reference level of said RPL10A.
  • the present invention relates to a (an) (in vitro) method of diagnosing lung cancer in a patient comprising the steps of:
  • RNP ribonucleoprotein
  • the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80% sequence identity thereto and a ribosomal protein L10A (RPL10A) in an extracellular fraction of blood of a patient, and
  • the present invention relates to a (an) (in vitro) method of diagnosing lung cancer in a patient comprising the steps of:
  • the above comparison allows to diagnose lung cancer in a patient, in particular in a patient suspected of suffering from lung cancer.
  • the patient may be diagnosed as suffering from lung cancer, i.e. being diseased, or as not suffering from lung cancer, i.e. being healthy.
  • the reference level may be any reference level which allows to determine whether the patient suffers from lung cancer or not.
  • the reference level may be obtained from (control) subjects (i.e. subjects different from the patient to be tested such as healthy subjects and/or subjects known to have lung cancer) or from the same patient. In the latter case, the patient may be retested for lung cancer, e.g. in the form of a longitudinal monitoring. It may be determined that the patient is now affected by lung cancer or still not affected by lung cancer.
  • the reference level is the level of the RNP complex, or the level of RPL10A determined empirically by measuring a number of reference extracellular fractions of blood of healthy subjects (not suffering from lung cancer), and/or subjects known to suffer from lung cancer.
  • the reference level is determined by measuring at least 2, at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000 at least 5000 reference extracellular fractions of blood of healthy subjects, and/or the reference level is determined by measuring at least 2, at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000 at least 5000 reference extracellular fractions of blood of subjects known to suffer from lung cancer.
  • the reference level is an average reference level. It is determined by measuring reference levels of control subjects (e.g. healthy subjects or subjects having lung cancer/known to have lung cancer) and calculating the “average” level (e.g. mean, median or modal value) thereof. It is preferred that the reference extracellular fraction of blood is from the same source (e.g. serum or plasma) than the extracellular fraction of blood obtained from the patient to be tested. It is further preferred that the reference level is obtained from control subjects (e.g. healthy subjects or subjects having lung cancer/known to have lung cancer) of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. adults or elderly) than the patient to be tested. Especially, the reference level represents an average value of the RNA molecule in a healthy population and/or the reference level represents an average value of the RNA molecule in a population of subjects having lung cancer/known to have lung cancer.
  • the “average” level e.g. mean, median or modal value
  • the reference level is the level of the RNP complex, or the level of RPL10A determined empirically by measuring a number of reference extracellular fractions of blood of healthy subjects (not suffering from lung cancer), wherein the level of the RNP complex, and/or the level of RPL10A above the reference level indicates that the patient suffers from lung cancer.
  • the level of the RNP complex and/or of RPL10A is at least 0.6-fold or 0.7- fold, more specifically at least 0.8-fold or 0.9-fold, even more specifically at least 1.2-fold or 1.5- fold, and still even more specifically at least 2.0-fold or 3.0-fold above the reference level.
  • the level of the RNP complex and/or of RPL10A is at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1.0-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9- fold, at least 2.0-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, or at least 3.0-fold above the reference level.
  • the level of a ribonucleoprotein (RNP) complex wherein the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80% sequence identity thereto and a ribosomal protein L10A (RPL10A) is/are determined in the method of diagnosing lung cancer in a patient.
  • the nucleotide sequence according to SEQ ID NO: 1 (CGCCGGUGAAAUACCACU) represents the “core sequence” which allows lung cancer diagnosis. This core sequence is also comprised in/encompassed by other nucleotide sequences which equally allow lung cancer diagnosis.
  • variants/precursors of the nucleotide sequence according to SEQ ID NO: 1 ( CGCCGGU GA A AU ACC ACU) as described herein have the following nucleotide sequences: GCCGCCGGUGAAAUACCACUAC (SEQ ID NO: 2) CGGGCCGCCGGUGAAAUACCACUAC (SEQ ID NO: 3),
  • ribonucleoprotein (RNP) complexes comprising any of the nucleotide sequences according to SEQ ID NO: 1 to SEQ ID NO: 5 and ribosomal protein L10A (RPL10A) may be determined in the method of the present invention.
  • RNP ribonucleoprotein
  • RPL10A ribosomal protein L10A
  • core sequence the nucleotide sequences according to SEQ ID NO: 2 to SEQ ID NO: 5 are covered.
  • the sequences according to SEQ ID NO: 1 to SEQ ID NO: 5 are preferably human sequences.
  • nucleotide sequences can be present in a 2’ -ortho-methylated or 2’-ortho- non-methylated state and optionally possess various 5’ ends, such as 5’ phosphorylation or 5’ hydroxyl and/or various 3’ ends, such as 3’ hydroxyl, 3’ phosphate or 3’, 2’ cyclic phosphate.
  • RNP ribonucleoprotein
  • RPL10A ribosomal protein L10A
  • the level of the RNP complex is determined by an immunoprecipitation-based assay.
  • the immunoprecipitation-based assay is, for example, an antibody-based pulldown of RPL10A followed by the detection of the co-immunoprecipitated RNA molecule.
  • Immunoprecipitation is a technique which allows the selectively isolation of a particular protein species from a sample.
  • the technique relies on antigen/antibody recognition (immuno) and the subsequent mass- or affinity-related isolation of the capture/target complex (precipitation). It is the precipitation of the target protein that is referenced by the name: Pull-down assay.
  • the pulldown assay for “pull down” RPL10A and, thereby the RNP complex comprising the RNA molecule involves the use of a matrix-associated antibody for RPL10A, and various wash and elution steps to ensure the specific preference for the targeted RPL10A. After isolation of the RNP complex via the RPL10A “pull down”, the RNA molecule is subsequently detected.
  • the RNA may be extracted from the complex via phenol- guanidyl chloride-based extraction (e.g. QIAzol). Otherwise, Protease K may be used to libereate the RNA molecule from the complex.
  • the detection of the co-immunoprecipitated RNA molecule is carried out by a Polymerase Chain Reaction (PCR) or next generation sequencing (NGS). More preferably, the PCR is selected from the group consisting of digital PCR, real-time PCR (quantitative PCR or qPCR), preferably TaqMan qPCR, multiplex PCR, nested PCR, high fidelity PR, fast PCR, hot start PCR, and GC-rich PCR.
  • RPL10A antibodies may be obtained from ChromoTek & Proteintech Germany, Ref# 16681-AP, OriGene Technologies GmbH, Ref# TA809520S, or NovusBio, Ref# NBP1-56304.
  • the level of the RNP complex is determined by an RNA- pulldown assay.
  • the RNA-pulldown assay is, for example, an antisense oligonucleotide-based pulldown of the RNA molecule followed by the detection of RPL10A.
  • RNA pulldown is a popular RNA-centric approach to study RNA-protein interaction.
  • Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence, in the present case to the RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1.
  • the antisense oligonucleotides bind the chosen sequence, in the present case the RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, in a process called hybridization.
  • antisense oligonucleotides can be used to target a specific, complementary RNA, in the present case the RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1.
  • the pull-down assay for “pull down” the RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1 and, thereby the RNP complex comprising RPL10A involves the use of a matrix-associated antisense oligonucleotide, and various wash and elution steps to ensure the specific preference for the targeted RNA molecule.
  • RPL10A is subsequently detected.
  • the detection of RPL10A is carried out by an antibody-based assay or a mass spectrometry-based assay.
  • the antibody-based assay is selected from the group consisting of an Enzyme-Linked Immunosorbent Assay (ELISA), an Immuno-Polymerase Chain Reaction (Immuno-PCR) assay, an Immuno-Next generation sequencing (Immuno-NGS) assay, and a Western Blot assay.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • Immuno-PCR Immuno-Polymerase Chain Reaction
  • Immuno-NGS Immuno-Next generation sequencing
  • Preferred antisense oligonucleotides for binding to/hybridizing with the RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1 are selected from the group consisting of DNA oligonucleotides containing a core sequence of 5’- AGTGGTATTTCACCGGCG-3’ (SEQ ID NO: 18), RNA oligonucleotides containing a core sequence 5’-AGUGGTAUUUCACCGGCG-3’ (SEQ ID NO: 19), and any of these oligonucleotides enhanced by any number of modifications such as 2’ -ortho methylation, locked- nucleid acids (LNA) and similar.
  • LNA locked- nucleid acids
  • the level of RPL10A is determined by an antibody-based assay or a mass spectrometry-based assay.
  • the antibody-based assay is selected from the group consisting of an Enzyme-Linked Immunosorbent Assay (ELISA), an Immuno-Polymerase Chain Reaction (Immuno-PCR) assay, an Immuno-Next generation sequencing (Immuno-NGS) assay, and a Western Blot assay, or the mass spectrometry-based assay is selected from the group consisting of a liquid chromatography-mass spectrometry (LC-MS) assay and fractionation followed by mass spectrometry (F/MS).
  • ELISA Enzyme-Linked Immunosorbent Assay
  • Immuno-PCR Immuno-Polymerase Chain Reaction
  • Immuno-NGS Immuno-Next generation sequencing
  • Western Blot assay or the mass spectrometry-based assay is selected from the group consisting of a liquid chromatography-mass spectrometry (LC-MS) assay and fractionation followed by mass spectrometry (F/MS).
  • LC-MS liquid chromatography
  • an RPL10A antibody is immobilized on a solid surface (microplate) and then complexed with RPL10A being part of the RNP complex comprising the RNA molecule. Subsequently, another antibody which binds RPL10A and which is linked to a reporter, e.g. reporter enzyme, is used. Detection is accomplished by measuring the activity of the reporter, e.g. reporter enzyme, via incubation with the appropriate substrate to produce a measurable product.
  • a reporter e.g. reporter enzyme
  • Coating/capture direct or indirect immobilization of antibodies to the surface of polystyrene microplate wells.
  • Plate blocking addition of irrelevant protein or other molecule to cover all unsaturated surfacebinding sites of the microplate wells.
  • Probing/detection incubation with proteins comprised in a sample that affinity-bind to the immobilized antibodies.
  • Signal measurement detection of the bound proteins, e.g. directly or via a secondary tag on another specific antibody.
  • Immuno-Polymerase Chain Reaction In an Immuno-Polymerase Chain Reaction (Immuno-PCR, IPCR) assay, for example, two RPL10A antibodies are allowed to bind to the RPL10A target. Each antibody bears an DNA oligonucleotide. Only proximity of the two oligonucleotides, what requires an antibody/protein bond, allows for oligonucleotide ligation and subsequent PCR detection.
  • the Immuno-Polymerase Chain Reaction (Immuno-PCR, IPCR) assay is the most sensitive technique for the detection of proteins and gains its uniqueness through the exponential amplification of a signal- generating nucleic acid intermediate attached to a protein target.
  • Immuno-Next generation sequencing (Immuno-NGS) assay, for example, two RPL10A antibodies are allowed to bind to the RPL10A target. Each antibody bears an DNA oligonucleotide. Only proximity of the two oligonucleotides, what requires an antibody/protein bond, allows for oligonucleotide ligation and subsequent detection via next generation sequencing.
  • a Western Blot assay for example, three elements are used to achieve the separation of a specific protein from a complex mixture: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize.
  • a primary antibody is used that recognizes and binds to RPL10A. Therefore, the electrophoresis membrane is washed in a solution containing primary antibody, before excess antibody is washed off. Subsequently, a secondary antibody is added which recognizes and binds to the primary antibody. The secondary antibody is visualized through various methods such as staining, immunofluorescence, and radioactivity allowing indirect detection of RPL10A.
  • the lung cancer e.g.
  • lung cancer of state I, II, III, or IV may be non- small-cell lung carcinoma (NSCLC) or small-cell lung carcinoma (SCLS).
  • NSCLC non- small-cell lung carcinoma
  • SCLS small-cell lung carcinoma
  • the lung cancer is early stage lung cancer, i.e. lung cancer of stage I.
  • the patient is preferably a mammal, more preferably a human.
  • the extracellular fraction of blood is selected from the group consisting of serum and plasma. More preferably, the plasma is stabilized by covalently-non-crosslinking or covalently-crosslinking chemical agents.
  • the present invention relates to the (in vitro) use of a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g.
  • RNP ribonucleoprotein
  • ribosomal protein L10A RPL10A
  • RPL10A ribosomal protein L10A
  • the ribonucleoprotein (RNP) complex wherein the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80% sequence identity thereto and a ribosomal protein L10A (RPL10A), and/or a ribosomal protein L10A (RPL10A) is/are part of/comprised in an extracellular fraction of blood of a patient.
  • the extracellular fraction of blood is selected from the group consisting of serum and plasma. More preferably, the plasma is stabilized by covalently- non-crosslinking or covalently-crosslinking chemical agents.
  • the patient is preferably a mammal, more preferably a human.
  • the lung cancer may be non- small-cell lung carcinoma (NSCLC) or small-cell lung carcinoma (SCLS).
  • NSCLC non- small-cell lung carcinoma
  • SCLS small-cell lung carcinoma
  • the lung cancer is early stage lung cancer, i.e. lung cancer of stage I.
  • RNA molecule comprising
  • nucleotide sequence that is a fragment of the nucleotide sequence according to (i), preferably a nucleotide sequence that is a fragment which is between 1 to 6, more preferably between 1 to 4, and most preferably between 1 to 2, e.g. 1, 2, 3, 4, 5, or 6, nucleotide(s) shorter than the nucleotide sequence according to (i), or
  • nucleotide sequence that has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the nucleotide sequence according to (i) or nucleotide sequence fragment according to (ii) is part of the RNP complex which is used to diagnose lung cancer.
  • a ribonucleoprotein (RNP) complex wherein the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80% sequence identity thereto and a ribosomal protein L10A (RPL10A) is used/detected for diagnosing lung cancer in a patient.
  • RNP ribonucleoprotein
  • nucleotide sequence according to SEQ ID NO: 1 represents the “core sequence” which allows lung cancer diagnosis.
  • This core sequence is also comprised in/encompassed by other nucleotide sequences which equally allow lung cancer diagnosis.
  • the variants/precursors of the nucleotide sequence according to SEQ ID NO: 1 ( CGCCGGU GA A AU ACC ACU) as described herein have the following nucleotide sequences: GCCGCCGGUGAAAUACCACUAC (SEQ ID NO: 2) CGGGCCGCCGGUGAAAUACCACUAC (SEQ ID NO: 3),
  • ribonucleoprotein (RNP) complexes comprising any of the nucleotide sequences according to SEQ ID NO: 1 to SEQ ID NO: 5 and ribosomal protein L10A (RPL10A) are used for lung cancer diagnosis.
  • RNP ribonucleoprotein
  • RPL10A ribosomal protein L10A
  • core sequence the nucleotide sequences according to SEQ ID NO: 1
  • SEQ ID NO: 2 to SEQ ID NO: 5 are covered.
  • the sequences according to SEQ ID NO: 1 to SEQ ID NO: 5 are preferably human sequences.
  • nucleotide sequences can be present in a 2’ -ortho-methylated or 2’-ortho- non-methylated state and optionally possess various 5’ ends, such as 5’ phosphorylation or 5’ hydroxyl and/or various 3’ ends, such as 3’ hydroxyl, 3’ phosphate or 3’, 2’ cyclic phosphate.
  • RNA molecule As to the preferred embodiments to detect the ribonucleoprotein (RNP) complex and/or the RNA molecule, it is referred to the first aspect of the present invention.
  • RNP ribonucleoprotein
  • the present invention relates to (the (in vitro) use of) a kit for diagnosing lung cancer, said kit comprising: means for determining the level of a a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g.
  • RNP ribonucleoprotein
  • RPL10A ribosomal protein L10A
  • RPL10A ribosomal protein L10A
  • the patient is preferably a mammal, more preferably a human.
  • the lung cancer may be non- small-cell lung carcinoma (NSCLC) or small-cell lung carcinoma (SCLS).
  • NSCLC non- small-cell lung carcinoma
  • SCLS small-cell lung carcinoma
  • the lung cancer is early stage lung cancer, i.e. lung cancer of stage I.
  • the extracellular fraction of blood is selected from the group consisting of serum and plasma. More preferably, the plasma is stabilized by covalently-non-crosslinking or covalently-crosslinking chemical agents.
  • RNA molecule comprising
  • nucleotide sequence that is a fragment of the nucleotide sequence according to (i), preferably a nucleotide sequence that is a fragment which is between 1 to 6, more preferably between 1 to 4, and most preferably between 1 to 2, e.g. 1, 2, 3, 4, 5, or 6, nucleotide(s) shorter than the nucleotide sequence according to (i), or
  • nucleotide sequence that has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the nucleotide sequence according to (i) or nucleotide sequence fragment according to (ii) is part of the RNP complex which is to be detected with the means as described herein.
  • the means are for determining the level of a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80% sequence identity thereto and a ribosomal protein L10A (RPL10A).
  • RNP ribonucleoprotein
  • nucleotide sequence according to SEQ ID NO: 1 represents the “core sequence” which allows lung cancer diagnosis.
  • This core sequence is also comprised in/encompassed by other nucleotide sequences which equally allow lung cancer diagnosis.
  • the variants/precursors of the nucleotide sequence according to SEQ ID NO: 1 ( CGCCGGU GA A AU ACC ACU) as described herein have the following nucleotide sequences: GCCGCCGGUGAAAUACCACUAC (SEQ ID NO: 2) CGGGCCGCCGGUGAAAUACCACUAC (SEQ ID NO: 3),
  • the means are for determining the level of ribonucleoprotein (RNP) complexes comprising any of the nucleotide sequences according to SEQ ID NO: 1 to SEQ ID NO: 5 and ribosomal protein L10A (RPL10A).
  • RNP ribonucleoprotein
  • RPL10A ribosomal protein L10A
  • nucleotide sequences can be present in a 2’ -ortho-methylated or 2’-ortho- non-methylated state and optionally possess various 5’ ends, such as 5’ phosphorylation or 5’ hydroxyl and/or various 3’ ends, such as 3’ hydroxyl, 3’ phosphate or 3’, 2’ cyclic phosphate.
  • the means for determining the level of ribosomal protein L10A comprise means for antibody-based detection of RPL10A, more preferably means for detecting RPL10A via an ELISA, an Immune-PCR assay, Immuno-NGS assay, and/or a Western Blot assay, and/or means for mass spectrometry-based detection of RPL10A.
  • the means for determining the level of ribosomal protein L10A (RPL10A) via ELISA may comprise one or more of the following: a solid surface (microplate) to which a ribosomal protein L10A (RPL10A) antibody (primary antibody) can bind, a ribosomal protein L10A (RPL10A) antibody (primary antibody) to which RPL10A can bind, another antibody which can bind RPL10A and which is linked to a reporter such as reporter enzyme (secondary antibody), and a substrate for the reporter such as the reporter enzyme.
  • a solid surface to which a ribosomal protein L10A (RPL10A) antibody (primary antibody) can bind
  • RPL10A) antibody primary antibody
  • another antibody which can bind RPL10A and which is linked to a reporter such as reporter enzyme (secondary antibody)
  • a substrate for the reporter such as the reporter enzyme
  • the means for determining the level of ribosomal protein L10A (RPL10A) via Immuno-Polymerase Chain Reaction (Immuno-PCR, IPCR) assay may comprise one or more of the following: two RPL10A antibodies which can bind to the RPL10A target, wherein each antibody bears an DNA oligonucleotide, means for carrying out oligonucleotide ligation (e.g. ligase buffer and ligase) and subsequent PCR detection (PCR puffer and DNA polymerase).
  • oligonucleotide ligation e.g. ligase buffer and ligase
  • subsequent PCR detection PCR puffer and DNA polymerase
  • the means for determining the level of ribosomal protein L10A (RPL10A) via Immuno-Next generation sequencing (Immuno-NGS) assay may comprise one or more of the following: two RPL10A antibodies which can bind to the RPL10A target, wherein each antibody bears an DNA oligonucleotide, means for carrying out oligonucleotide ligation (e.g. ligase buffer and ligase) and subsequent next generation sequencing.
  • two RPL10A antibodies which can bind to the RPL10A target, wherein each antibody bears an DNA oligonucleotide
  • means for carrying out oligonucleotide ligation e.g. ligase buffer and ligase
  • the means for determining the level of ribosomal protein L10A (RPL10A) via Western Blot assay may comprise one or more of the following: an antibody which can bind RPL10A (primary antibody), another antibody which can bind the primary antibody (secondary antibody).
  • the secondary antibody can further comprise a reporter, e.g. a reporter which can be visualized through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of RPL10A.
  • the means for determining the level of the ribonucleoprotein (RNP) complex comprising an RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1, a fragment thereof, or a sequence having at least 80% sequence identity thereto and a ribosomal protein L10A (RPL10A) comprise means for an immunoprecipitation-based assay to detect the complex, or means for an RNA-pulldown assay to detect the complex.
  • the means for determining the level of the complex via an immunoprecipitationbased assay comprise one or more of the following: a matrix to which an antibody for RPL10A can bind, an antibody for RPL10A, means for detecting the co-immunoprecipitated RNA molecule such as means for carrying out a Polymerase Chain Reaction (PCR) or next generation sequencing (NGS), and optionally a washing buffer, an elution buffer and/or a protease.
  • PCR Polymerase Chain Reaction
  • NGS next generation sequencing
  • the means for determining the level of the complex via an RNA-pulldown assay comprise one or more of the following: an antisense oligonucleotide, specifically a matrix- associated antisense oligonucleotide, for binding the RNA molecule, means for detecting the co- immunoprecipitated RPL10A, such as means for carrying out an antibody-based assay or a mass spectrometry-based assay, and optionally a washing buffer and/or an elution buffer.
  • an antisense oligonucleotide specifically a matrix- associated antisense oligonucleotide, for binding the RNA molecule
  • means for detecting the co- immunoprecipitated RPL10A such as means for carrying out an antibody-based assay or a mass spectrometry-based assay, and optionally a washing buffer and/or an elution buffer.
  • RPL10A antibodies may be obtained from ChromoTek & Proteintech Germany, Ref# 16681-AP, OriGene Technologies GmbH, Ref# TA809520S, or NovusBio, Ref# NBP1-56304, and/or antisense oligonucleotides for binding to/hybridizing with the RNA molecule comprising a nucleotide sequence according to SEQ ID NO: 1 are selected from the group consisting of DNA oligonucleotides containing a core sequence of 5’-AGTGGTATTTCACCGGCG-3’ (SEQ ID NO: 18), RNA oligonucleotides containing a core sequence 5’-AGUGGTAUUUCACCGGCG-3’ (SEQ ID NO: 19), and any of these oligonucleotides enhanced by any number of modifications such as 2’ -ortho methylation, locked- nucleid acids (LNA) and similar.
  • the kit may further comprise a container, and/or a data carrier.
  • the data carrier may be a non-electronical data carrier, e.g. a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronical data carrier.
  • the access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized database.
  • the access code may also allow access to an application software that causes a computer to perform tasks for computer users or a mobile app which is a software designed to run on smartphones and other mobile devices.
  • Said data carrier may further comprise at least one reference, e.g. reference level(s) of the complex or protein determined herein and/or cut-off score(s).
  • at least one reference e.g. reference level(s) of the complex or protein determined herein and/or cut-off score(s)
  • said at least one reference e.g. reference level(s) of the complex or protein determined herein and/or cut-off score(s)
  • the kit is useful for conducting the method according to the first aspect.
  • the kit comprises instructions on how to carry out the method according to the first aspect.
  • Figure 2 Position of miLung#l and two of its precursors is indicated in the context of the mature 28S rRNA sequence (SEQ ID NO: 2).
  • Figure 6 Digital PCR expression of miLung#l in RNA samples isolated from human serum that was control-treated or treated with RNaseA, or proteinase K and RNaseA, respectively. Two sets of treatments were measured in duplicates.
  • Figure 7 Digital PCR expression of miLung#l in RNA samples isolated from human peripheral blood (donor#5) using various preservation tubes, either processed freshly or as frozen material.
  • the cell- free fractions were either control-treated, treated with RNaseA, or proteinase K and RNaseA, respectively. Duplicated measurements were performed.
  • Figure 8 Digital PCR expression of miLung#l in RNA samples isolated from cancer patient serum that was control-treated or treated with RNaseA, or proteinase K and RNaseA, respectively. Two sets of treatments were measured in duplicates.
  • Figure 9 An image of RPL10A protein and miLung#l RNA as derived from the cryo electron microscopy (ProteinDataBank accession code 5T2C).
  • Figure 10 Western blotting image of immuno-precipitation experiment. * indicates unspecific signal.
  • Figure 11 Digital PCR measurements of miLung#l biomarker and two unrelated rRNA fragments as enriched by immunoprecipitation (IP) from conditioned media (miLung#l / rRNA 2718-2738: Replicate 1 - left, Replicate 2 - right, miLung#l / rRNA 2503-2523: Replicate 1 - left, Replicate 2 - right).
  • IP immunoprecipitation
  • HCC-827 and A-549 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). HCC-827 were cultured in RPMI medium supplemented with 20% fetal bovine serum. A-549 were cultured in DMEM medium supplemented with 10% fetal bovine serum. Both media contained 1% penicillin/streptomycin.
  • NGS Next generation sequencing
  • RNAs are extended using adapter molecules and converted to complementary DNA (cDNA).
  • cDNAs are amplified using PCR reaction and the products are loaded on flow cell that captures the cDNA molecules and determines their sequence (and thus the sequence of the small RNA) in a massive parallel fashion.
  • Localy amplified cDNA molecules on flow-cell form clusters and the determination of the number of clusters for a given cDNA molecule provides relative measurement of its expression, often denoted as reads per million (RPM) of all clusters or all reads.
  • RPM reads per million
  • RNA elution or 100 ng of RNA was used for 3’ adapter ligation reaction as outlined below.
  • Pre-adenylated DNA linker sequence 5’-AACTGTAGGCACCATCAAT-3’ (SEQ ID NO: 6)
  • RNA adapter sequence 5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’ (SEQ ID NO: 7) The reaction was held 30 min at 28°C followed by 20 min at 65°C. Then was the reaction cooled to 4°C. To the previous reaction, following components were added:
  • the reaction was denatured at 85°C for 2 min, 2 min at 75°C, 2 min at 70°C, 2 min at 65°C, 2 min at 60°C, 2 min at 55°C, 5 min at 37°C and finally 25°C for 5 min. Then the reaction was placed on ice and following components were added:
  • the sequence of the RT primer DNA oligo is as follows (where “+” denotes locked nucleic acid,
  • N is any deoxyribonucleotide
  • the reaction was incubated at 50°C for 60 min and then 15 min at 70°C. The reaction was then placed on ice.
  • the cDNA was purified using Omega MagBind pure NGS beads (Omega biotek) as recommended by the vendor. 2.5x ratio of the beads to the cDNA reaction volume was used.
  • the cDNA was eluted in 25 pl of 10 mM Tris-HCl, pH 8.5.
  • cDNA was used for PCR using following components:
  • the sequence of the indexing primers were as follows (“*” denotes phosphortioate bond, while “N” denotes 8 nucleotide index sequence unique for each primer):
  • the PCR reaction conditions were as follows:
  • the PCR product was purified using Omega MagBind pure NGS beads as recommended by the vendor.
  • a 0.9x ratio of the beads to the PCR reaction volume was used and supernatant was used for second binding reaction with 1.8x ratio of beads to supernatant.
  • the DNA was eluted in 13 pl of 10 mM Tris-HCl, pH 8.5.
  • DNA concentration was determined using QuantIT (Thermo fisher scientific). Equimolar pool of DNA libraries was prepared and loaded as 500 pM concentration on NextSeq2000 P3 50 cycles cartridge (Illumina). Custom index 2 primer was used for sequencing (sequence: 5’- GATCGTCGGACTGTAGAACTCTGAACGTGT-3’ (SEQ ID NO: 11)).
  • sequence files (fasta) were preprocessed by trimming the 3 ’adapter sequence. Read count matrix was established and RPM values were calculated.
  • HBDx digital PCR To measure expression of a given small RNAs in the biological samples, a HBDx digital PCR method is used. Shortly, the RNA are extended using anti-complementary adapter molecules and converted to complementary DNA (cDNA). cDNAs are amplified using PCR reaction and the products are detected using defined fluorescently labelled probes in thousands of partitions of a larger PCR mixture. Because only some partitions will be positive, their ratio to the total number of partitions (positive and negative) is proportional to the orginal number of molecules in the biological RNA sample, enabling thus absolute quantification of the RNA expression.
  • cDNA complementary DNA
  • adapters are denatured and renatured such that they form required stem loop-like structures.
  • the source RNA is denatured separately, treated with the polynucleotide kinase (to restore the 5’ phosphate), mixed with adapters and used for the ligation by T4 RNA ligase 2.
  • RT primer aligning to the 3’ adapter is used for the cDNA production.
  • diluted cDNA is used for the digital PCR with a reverse primer aligning to the 5’ end of first strand cDNA and forward primer complementary to the 3’ end of the first strand cDNA.
  • a TaqMan probe is used for the detection of the specific signal in digital PCR.
  • RT primer 5’-CTCAGTGCGAATACCTCGGACCCTGCACTGAGGTAGT (SEQ ID NO: 14)
  • Taqman Probe 5’-/6-FAM/TGAGGTAGTGGTATTTCACCGGCGGCCGT/BHQ-l/ (SEQ ID NO:
  • Reverse primer 3’-GTGCGAATACCTCGGACC (SEQ ID NO: 17) RESULTS
  • RNA can be resistant to the RNase by being included in a complex with other molecules that “protect” the RNA.
  • molecules are (RNA-binding) proteins that form a complex with RNA; such complex is termed ribonucleoprotein complex, or “RNP”.
  • RNP ribonucleoprotein complex
  • miLung#l RNP detection techniques can be used for the diagnosis of lung cancer. Especially techniques that employ (simultaneous) detection of RNP protein(s) using antibodies raised against these protein(s) and/or detection of miLung#l using anti-complementary oligonucleotides may enable sensitive and specific detection of miLung#l RNP and can be used for (early) lung cancer detection.
  • miLung#l refers to the RNA molecule having a nucleotide sequence according to SEQ ID NO: 2 ( GCCGCCGGU GA A AU ACC ACU AC) .
  • SEQ ID NO: 2 GCCGCCGGU GA A AU ACC ACU AC
  • the RNP complex detection in a cell-free fraction of blood of a patient can include dilution of the biological material, flow of the diluted material and immobilization of the RNP complex on a solid support using an antibody against RPL10A attached thereto, washing and subsequent detection of the RNA molecule in the complex using anti-complementary flurophore labelled probes. Upregulation of the complex in the cell-free fraction of blood of a patient compared to a healthy control sample is indicative for lung cancer.
  • the RPL10A detection in a cell-free fraction of blood of a patient can include dilution of the biological material, flow of the diluted material and immobilization of the RPL10A on a solid support using an antibody against RPL10A attached thereto. Subsequently, another antibody which binds RPL10A and which is linked to a reporter, e.g. reporter enzyme, may be used. Detection is accomplished by measuring the activity of the reporter, e.g. reporter enzyme, via incubation with the appropriate substrate to produce a measurable product. Up-regulation of RPL10A in the cell-free fraction of blood of a patient compared to a healthy control sample is indicative for lung cancer.
  • HCC-827 cells expressing HA tag and Prescission cleavage site at the N-terminus of RPL10A were washed 2x with PBS and cultured overnight in growth medium without fetal calf serum.
  • the concentrated medium was diluted with lysis buffer to 1 ml, such that the final concentration of lysis buffer components were: 50 mM NaCl, 10 mM Tris-HCl pH7.5, 0.5 rnM EDTA, 0.05 mM EGTA, 1 % Triton-XlOO, lx protease inhibitor cocktail and 10 units of murine RNase inhibitor.
  • IP immunoprecipitation
  • Second tube was used for competitive elution by HA-peptide with final concentration of 2 mg/ml.
  • Third tube was used for protease cleavage using 2 pl Prescission protease in 50 pl total cleavage buffer (50mM TRIS-HC1, pH7.0, 150mM NaCl, ImM EDTA pH8.0, ImM DTT) for 4 hours at 4°C.
  • 50 pl total cleavage buffer 50mM TRIS-HC1, pH7.0, 150mM NaCl, ImM EDTA pH8.0, ImM DTT
  • Protein samples were mixed with 4x sample buffer, boiled for 3 min at 90°C, loaded on 4-12% denaturating SDS-PAGE gel, and blotted to nitrocellulose membrane.
  • Membrane was blocked for 1 hour in 5% milk in PBS, 0,05% Tween- 20.
  • Anti-RPLIO antibody (#A305-061A) was used in 1:8000 concentration in 5% milk in PBS-T overnight at 4°C. After 3 washes with PBS-T, the membrane was stained with anti-rabbit HPR-conjugated secondary antibody for 1 hour at room temperature in 5% milk in PBS-T. The membrane was washed 3 times in PBS-T and developed using ECL chemistry.
  • RNA samples isolated from input and IP samples were used for digital PCR detection using Dumbbell PCR protocol as described in, specifically in the experimental section of, WO 2023/016938.
  • the adapters and probes used for additional 28S rRNA fragments are outlined in Table 1 below.
  • miLung#l-contatining particles may involve other molecules than proteins only, such as glycoproteins or double-layered membranes.
  • the additional 28S rRNA fragment 2718-2738 has the sequence according to SEQ ID NO: 20 and the additional 28S rRNA fragment 2503-2523 has the seqeunce according to SEQ ID NO: 21 ( Figure 11).
  • rRNA 2718-2738 SEQ ID NO: 20: rUrCrCrGrGrUrGrArGrCrUrCrUrCrGrCrUrGrGrCrC rRNA 2503-2523 (SEQ ID NO: 21): rGrCrCrGrArUrCrGi ArAi ArGrGrGrArGrUrCrGrGrGrGrG
  • RPL10A protein is responsible for the binding and protection of miLung#l in the conditioned growth medium of cancer cell line HCC-827.
  • miLung#l biomarker is stabilized in peripheral blood by interaction with (at least) RPL10A protein, and protection effect of this interaction is enhanced in the cancer patient serum samples as compared to healthy donors.

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Abstract

La présente invention concerne une méthode de diagnostic du cancer du poumon chez un patient. La présente invention concerne en outre un kit pour mettre en oeuvre ce procédé.
PCT/EP2024/074430 2023-09-05 2024-09-02 Complexe arn-protéine en tant que nouveau biomarqueur pour le cancer du poumon Pending WO2025051668A1 (fr)

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