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WO2025109033A1 - Procédé pour identifier si un sujet présente un risque de développer un cancer du poumon - Google Patents

Procédé pour identifier si un sujet présente un risque de développer un cancer du poumon Download PDF

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Publication number
WO2025109033A1
WO2025109033A1 PCT/EP2024/083042 EP2024083042W WO2025109033A1 WO 2025109033 A1 WO2025109033 A1 WO 2025109033A1 EP 2024083042 W EP2024083042 W EP 2024083042W WO 2025109033 A1 WO2025109033 A1 WO 2025109033A1
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cfdna
lung cancer
subject
sample
nasal
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Andrea STEPHANY
Pablo LARA ARENAS
Roberta GATTA
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Oncoswab GmbH
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Oncoswab 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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

  • Lung cancer is by far the leading cause of cancer death in the US, accounting for about 1 in 5 of all cancer deaths.
  • lung cancer There are 2 main types of lung cancer including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Most people diagnosed with lung cancer are 65 or older. Early lung cancer often has no symptoms and can only be detected by medical imaging such as low dose computed tomography (LDCT).
  • LDCT low dose computed tomography
  • DNA can be extracted from tumor tissue sample, or cell-free DNA (cfDNA) can be extracted from plasma sample for which real time PCR can be performed to detect variants of biomarkers.
  • NGS next generation sequencing
  • the present invention relates to a method for identifying if a subject is at risk of developing lung cancer, the method comprising detecting in at least one sample from said subject at least one or more cell free DNA (cfDNA) being fragments of target genes selected from the group comprising CDH13, CHD1 , DAPK, RASSF1 , SEPT9, ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1 , MET, NRAS, PIK3CA, ROS1 and/or TP53.
  • cfDNA cell free DNA
  • a subject at risk of developing lung cancer is a subject at risk of developing and/or having a lung cancer.
  • Early-stage lung cancer may develop or progress further over time.
  • Lung cancer can be detected at all stages of development, at early stage or at other stages of development by the method of the invention.
  • the invention relates also to a method for identifying if a subject is at risk of having lung cancer.
  • Circulating free nucleic acids such as circulating free DNA (cfDNA) or RNA (cfRNA) are present in biological fluids independent of the cells.
  • cell free DNA are circulating free DNA found in different body fluids, both in healthy and not healthy subjects.
  • cfDNA is released from cells into the circulatory system throughout the body and can be found for example in plasma, cerebral spinal fluid (CSF), pleural fluid, urine, nasal fluid, and saliva.
  • CSF cerebral spinal fluid
  • pleural fluid pleural fluid
  • urine circulating tumor DNA
  • ctDNA circulating tumor DNA
  • the method comprises detecting in at least one nasal sample from said subject at least one or more cell free DNA (cfDNA) that are fragments of target genes comprising ALK, EGFR, MAP2K1 , PIK3CA and TP53.
  • cfDNA cell free DNA
  • the method comprises the analysis of the target genes comprising ALK, EGFR, MAP2K1 , PIK3CA, TP53, KRAS, MET, NRAS, CDH13, CHD1 , DAPK, RASSF1 , SEPT9, BRAF, ERBB2, and ROS1 .
  • the analysis can be carried out for example by PCR analysis or NGS analysis.
  • the analysis of the target genes ALK, EGFR, MAP2K1 , PIK3CA and TP53 has been made using at least one nasal sample per subject.
  • the method is very reliable as at least one or more cfDNA/ctDNA being fragments of the target genes ALK, EGFR, MAP2K1 , PIK3CA and TP53 were found in 6 out of 7 lung cancer patients using nasal samples, whereas no ctDNA were found in healthy patients ( Figure 3 and example 8).
  • the efficacy of the method is at least 85%. This result is unexpected as the nasal cavity lacks a direct connection to tumor lung tissue.
  • the method of the present invention through the detection of cfDNA/ctDNA can be performed using samples obtained from subjects through a non- invasive procedure using a swab.
  • the collection of the sample may be done by rotating a swab on the nasal mucosa, or by rubbing the nasopharyngeal cavity and/or oropharynx.
  • the collection of nasal samples may be done by gently and slowly rotating a swab on the nasal mucosa of the anterior nare zone.
  • the nasal sample collection is easy, safety and cost-effective.
  • the target genes are selected from the group comprising ALK, EGFR, MAP2K1 , PIK3CA, TP53, KRAS, MET, and NRAS.
  • ctDNA have been found in nasal samples from lung cancer patients.
  • a large number of lung cancer mutations (68 mutations) were found in cfDNA/ctDNA being fragments of target genes selected from ALK, EGFR, KRAS, MAP2K1 , MET, NRAS, PIK3CA and TP53 (Table 8).
  • the target genes are selected from the group comprising ALK, EGFR, MAP2K1 , PIK3CA, TP53, KRAS, MET, NRAS, CDH13, CHD1 , DAPK, RASSF1 , SEPT9, BRAF, ERBB2, and ROS1.
  • the method of the invention may further comprises analyzing the presence or absence of genetic variants of the at least one or more cfDNA when cfDNA being fragments of target genes selected from the group comprising CDH13, CHD1 , DAPK, RASSF1 , SEPT9, ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1 , MET, NRAS, PIK3CA, ROS1 and/or TP53 have been detected in a sample obtained from the subject.
  • the method of the invention may further comprises analyzing the presence or absence of genetic variants of the at least one or more cfDNA when cfDNA being fragments of target genes selected from the group comprising ALK, EGFR, MAP2K1 , PIK3CA and TP53 have been detected in a sample obtained from the subject.
  • a nasal sample can be combined with any other type of samples from body fluids such as saliva, plasma, cerebral spinal fluid (CSF), pleural fluid, urine, sputum, stool, and/or seminal fluid.
  • body fluids such as saliva, plasma, cerebral spinal fluid (CSF), pleural fluid, urine, sputum, stool, and/or seminal fluid.
  • the method may comprise the step of combining at least two samples independently selected from nasal samples and/or salivary samples obtained from the subject.
  • the method comprises the step of combining at least two, three, four, five, six, seven, eight, nine, ten or more samples from nasal samples and/or a salivary sample. More preferably, the method comprises the step of combining at least two, three, four, five, six, seven, eight, nine, ten or more samples from nasal samples.
  • the method may comprise detecting in at least two nasal samples obtained from the subject at different time points at least one or more cfDNA being fragments of target genes selected from the group comprising CDH13, CHD1 , DAPK, RASSF1 , SEPT9, ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1 , MET, NRAS, PIK3CA, ROS1 and/or TP53.
  • the target genes are selected from the group comprising ALK, EGFR, MAP2K1 , PIK3CA and TP53.
  • the method comprises the step of monitoring over time the presence of cfDNA/ctDNA in at least two nasal samples from said subject at different time points during one, two, three, four, five, six, seven, eight, nine, ten or more weeks.
  • the detection of the at least one or more cfDNA may be determined by polymerase chain reaction (PCR) analysis.
  • PCR polymerase chain reaction
  • PCR analysis can be performed by digital droplet PCR (ddPCR), and/or multiplex PCR.
  • ddPCR digital droplet PCR
  • multiplex PCR analysis refers to the amplification of several cfDNA sequences simultaneously using multiple primers in one reaction, in which, preferably the multiplex PCR is carried out using primers for amplification only of the genetic variants of cfDNA (i.e. the fraction of cfDNA being ctDNA) comprising targeted mutations.
  • ddPCR analysis can be used to identify and quantify a targeted mutation (a hotspot) in the target genes such as those determined in the present invention (table 8 or table 1 ) with very high sensitivity.
  • the PCR analysis is a digital droplet PCR (ddPCR) analysis.
  • ddPCR digital droplet PCR
  • the PCR analysis comprises using probes hybridizing the genetic variants of the at least one or more cfDNA being fragments of the target genes ALK, EGFR, MAP2K1 , PIK3CA, TP53, KRAS, MET, NRAS, CDH13, CHD1 , DAPK, RASSF1 , SEPT9, BRAF, ERBB2, and/or ROS1.
  • the genetic variants of the said at least one or more cfDNA are selected from the genetic variants described in table 1 and/or table 8.
  • the method of the invention further comprises analyzing the presence of genetic variants of the at least one or more cfDNA, for example by nucleic acid sequencing analysis.
  • nucleic acid sequencing analysis includes methods for determining the sequence of nucleic acids selected from DNA and/or RNA.
  • the method further comprises a nucleic acid sequencing analysis, preferably the nucleic acid sequencing analysis is next-generation sequencing analysis (NGS).
  • NGS next-generation sequencing analysis
  • genetic variants of cfDNA include substitutions, insertions, deletions, gene rearrangements, exon skipping, fusions and copy number alterations in cfDNA and/or cfRNA.
  • genetic variants of cfDNA are ctDNA and the presence of at least one or more ctDNA in a sample from a subject indicates that said subject has an increased risk of having and/or developing a cancer.
  • the method of the invention may be carried out in combination with low dose computed tomography (LDCT), prior or after LDCT.
  • LDCT low dose computed tomography
  • a low dose computed tomography (LDCT) may be carried out to confirm the prognostic made by the method of the invention and establish a diagnosis.
  • each lung cancer patient has a specific profile (figure 3).
  • the analysis of nasal samples from said patients shows the presence of at least one, two, or three cfDNA being fragments of target genes independently selected from the group comprising ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1 , MET, NRAS, PIK3CA, ROS1 and TP53 and genetic variants of these cfDNA (ctDNA) have been found in said nasal samples.
  • the method may further comprise the step of detecting the presence or absence of 16S rRNA genes in said sample, preferably, in a nasal sample.
  • the method may further comprise the step of detecting and/or analyzing the presence or absence of genetic variants of 16S rRNA genes.
  • bacterial species contain more than one ribosomal RNA operon copy in their genomes, with some species containing up to 15 such copies.
  • the presence of genetic variants of 16S rRNA (rrs) genes indicates the presence of microorganisms in a sample.
  • the analysis of the nasal microbiome profile of patients with lung cancer in comparison with the microbiome profile of healthy subjects show the presence of multiple bacteria selected from the phylum Actinobacteriota, Bacteroidota, Firmicutes, and/or Proteobacteria.
  • the relative abundance of some bacteria such as Corynebacterium, Dolosigranulum or Moraxella was found higher than in samples from healthy subject (figure 5A and 5B).
  • the presence of genetic variants of the at least one or more cfDNA and/or 16S rRNA genes indicates that said subject has an increased risk of developing and/or having lung cancer.
  • the invention in another embodiment, relates to a method for identifying if a subject is at risk of developing lung cancer, the method comprising detecting in at least one sample from said subject at least one or more cell free DNA (cfDNA) being fragments of target genes selected from the group comprising CDH13, CHD1 , DAPK, RASSF1 , SEPT9, ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1 , MET, NRAS, PIK3CA, ROS1 and/or TP53, wherein the detection of at least one or more cfDNA may be determined by fast aneuploidy screening test sequencing system (Fast-seq).
  • cfDNA cell free DNA
  • Fast-seq may be used to analyze somatic copy number alterations (SCNA) which are present in cfDNA of lung cancer subjects and estimate ctDNA level. Following positive detection by Fast-seq of cfDNA/ctDNA, samples are analyzed subsequently by carrying out a nucleic acid sequencing analysis to detect variants in said cfDNA.
  • SCNA somatic copy number alterations
  • the method may further comprise the step of detecting aneuploidy levels in circulating free DNA (cfDNA) through genome sequencing, either before, during, or after treatment.
  • cfDNA circulating free DNA
  • the detection of tumor-derived aneuploidy in the at least one or more cfDNA/ctDNA indicates that said subject has an increased risk of developing lung cancer.
  • the invention relates to a method for identifying if a subject is at risk of developing lung cancer, the method comprising detecting in at least one sample from said subject at least one or more cell free DNA (cfDNA) being fragments of target genes selected from the group comprising CDH13, CHD1 , DAPK, RASSF1 , SEPT9, ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1 , MET, NRAS, PIK3CA, ROS1 and/or TP53, wherein the detection of at least one or more cfDNA is determined by PCR analysis and/or fast aneuploidy screening test sequencing system (Fast-seq).
  • cfDNA cell free DNA
  • the method may also comprise a step of detecting in at least one sample from said subject at least one or more cfRNA of target genes selected from the group comprising ALK, ROS1 , RET, NTRK, and/or MET.
  • the present invention also relates to a method for identifying if a subject is at risk of developing lung cancer disease, the method comprising i) detecting in at least one sample from said subject at least one or more cell free DNA (cfDNA) being fragments of target genes independently selected from the group comprising CDH13, CHD1 , DAPK, RASSF1 , SEPT9, ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1 , MET, NRAS, PIK3CA, ROS1 and/or TP53; and ii) determining clinical risk factors of said subject.
  • cfDNA cell free DNA
  • the method further comprises analyzing the presence or absence of genetic variants of the at least one or more cfDNA.
  • Clinical risk factors are for example selected from the group comprising age (years), gender, race or ethnic group, education, bmi (kg/m2), copd, emphysema, personal history of cancer, family history of lung cancer, personal history of pneumonia, smoking status (current smoker, former smoker), smoking duration (years), smoking intensity (cigarettes per day), pack-years of smoking, years since cessation, breathing upon exertion, cough intensity, changes in voice, flu or pneumonia within the past 2 year, exposure to asbestos or other chemicals, current residence, physical activity, exposures (coronary heart disease, myocardial infarction, stroke, diabetes mellitus, chronic kidney disease, arthritis, asthma), personal history of cancer.
  • the method further comprises the determination of clinical risk factors of said subject, said clinical risk factors are selected from the group comprising smoking status, age, gender, BMI, personal history of cancer, family history of lung cancer, personal history of pneumonia, and/or physical activity.
  • the method of the invention provides a comprehensive lung cancer management including in addition to the detection of the presence or absence of genetic variants of the at least one or more cfDNA, a risk assessment of high-risk population based on clinical risk factors.
  • lung cancer may be selected from non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, incidental pulmonary nodules related lung cancer and/or mesothelioma.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • bronchogenic carcinoma alveolar carcinoma
  • bronchial adenoma small cell lung cancer
  • SCLC small cell lung cancer
  • bronchogenic carcinoma alveolar carcinoma
  • bronchial adenoma sarcoma
  • lymphoma chondromatous hamartoma
  • incidental pulmonary nodules related lung cancer and/or mesothelioma may be adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, large cell carcinoma and sarcomatoid.
  • the method of the invention may further comprise the step of treating the subject with at least one anticancer agent or a combination thereof based on the detected presence of genetic variants of the at least one or more cfDNA.
  • the method of the invention makes it possible to identify mutations relevant to a specific treatment or therapy, which improves the precision and effectiveness of therapeutic options.
  • the treatment is tailored to a lung cancer patient in correlation with the type of lung cancer (NSCLC, SCLC, bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, and mesothelioma), and genetic variants of target genes/cfDNA detected in that patient.
  • NSCLC type of lung cancer
  • SCLC bronchogenic carcinoma
  • alveolar carcinoma bronchial adenoma
  • sarcoma sarcoma
  • lymphoma chondromatous hamartoma
  • mesothelioma mesothelioma
  • patients under treatment with a known mutation in the target genes of the invention can be monitored with the method of the invention using nasal fluid samples, for example by carrying out a PCR reaction such as ddPCR to follow the patient over time for their response to treatment.
  • a PCR reaction such as ddPCR
  • the method of the invention provides a comprehensive lung cancer management of lung cancer patients at all stages of the disease to follow the efficacy of a treatment and remission.
  • the method may further comprise the step of monitoring over time the response of the subject to the treatment with at least one anticancer agent.
  • Specific probes can also be used for detecting genetic variants of cfDNA being fragments of target genes selected from the group comprising ALK, EGFR, MAP2K1 , PIK3CA, TP53, KRAS, MET, NRAS, CDH13, CHD1 , DAPK, RASSF1 , SEPT9, BRAF, ERBB2, and ROS1.
  • the samples are centrifuged and mixed AMPurecourt beads for purification and analyzed by NGS.
  • a single multiplex PCR amplification is carried out to amplify using primer sequences as follows:
  • PCR amplifications consist of 9 pl DNA, 10 pl KAPA2G Robust DNA Polymerase (KapaBiosystems) and 1 pl of primers (0.2 pM final for each primer).
  • the PCR program is 95°C for 3 minutes, 35 x 95°C for 15s, 60°C for 15s, 72°C for 15s followed by 3 min at 72°C.
  • DNA sequencing data, identifying genetic variants, and infer disease progression is generated by the Ion Torrent sequencer or equivalent software.
  • Unaligned bam files which contain the raw sequencing data, are processed to align the sequences to the human reference genome (GRCh37/hg19), which provides a framework for identifying differences between the sample DNA and the reference genome.
  • variant calling is performed using the Ion Torrent-specific software (Torrent Variant Caller (TVC)-5.0.2.) which analyses the sequences to identify genetic variants.
  • Variant interpretation is performed using Genetic Assistant software, which assigns functional prediction, conservation scores, and disease-associated information to each variant based on relevant databases and literature. Integrative Genomics Viewer is used to visually inspect the variants and validate their presence in the samples.
  • Quantitative trait loci analyses are used to create directed links relating genetic variation in DNA mutations and transcriptional activity of pathways in cancer progression and recurrence.
  • the genomic profiling of the microbiome present in the nose is also explored using rRNA gene sequencing targeting the V3-V4 hypervariable regions of the 16S rRNA gene (primers 341 F and 785R). The 5’ ends of the primers for each sample will be tagged with specific barcodes, followed by purification of the amplicons and quantification.
  • Library preparation and sequencing will be performed using the standard instructions of the 16S Metagenomic Sequencing Library Preparation protocol (HluminaTM, Inc., San Diego, CA, United States). The measurements show the relative/absolute abundance of bacteria species in a sample in correlation with lung cancer.
  • the presence of 16S rRNA gene, and thus genetic variants of 16S rRNA gene indicates that said subject has an increased risk of developing a lung cancer.
  • Example 3 Biomarkers for lung cancer
  • over 200 potential cancer biomarkers have been detected in nasal fluids such as those described in Table 4.
  • Biomarkers found in saliva are shown in Table 5 below.
  • Example 4 Biomarkers for lung cancer - Blood sample.
  • biomarkers from blood can be measured in the nasal fluids.
  • Table 6 Biomarkers for lung cancer from blood sample
  • Example 5 Screening process to identify if a person at risk of developing a lung cancer
  • Figure 1 illustrates the process of identifying a person at risk of developing a lung cancer.
  • a nasal swab sample is obtained from a person to identify whether the person is at risk of developing a lung cancer.
  • DNA extraction from nasal swab samples is performed using the method as described in example 1 to prepare a library.
  • a PCR reaction using the protocol as described herein is performed with the sample containing cell free DNA (cfDNA).
  • sequencing analysis such as Next Generation Sequencing is carried out to identify the presence of biomarkers specific for lung cancer or somatic copy number alterations (SCNA), which are present in lung cancer.
  • the absence of amplification of cfDNA following PCR reaction is indicative that the person is not at risk of developing a lung cancer.
  • 16S-RNA seq is performed to analyze the nasal microbiome.
  • the microbiome can identify smokers from non-smokers as well as correlate with the risk of lung cancer.
  • DNA samples extracted from nasal swab sample are submitted to Fast Aneuploidy Screening Test-Sequencing System (Fast-seq). It is a faster and cost-effective method that simplifies the analysis of the genome by targeting repetitive fragments from different locations in the genome using a single primer pair (SEQ ID NO: 61 -87). With this alternative method, high throughput and decreased cost can be achieved by replacing laborious sequencing library preparation steps with PCR employing a single primer pair designed to amplify a discrete subset of repeated regions, which are present in lung cancer and involve alterations in a large portion of the cancer genome.
  • Fast-seq Fast Aneuploidy Screening Test-Sequencing System
  • Amplicon libraries targeting LINE-1 (L1 ) elements are prepared from 1 ng of cell-free DNA (cfDNA).
  • Target-specific L1 primers and Phusion Hot Start II Polymerase are utilized following the method outlined in a previous publication (https://doi.Org/10.1002/1878-0261 .13196).
  • the resulting PCR products undergo purification using AMPure Beads (Beckman Coulter) and serve as templates for a second PCR.
  • sequencing adaptors and sample-specific indexes are incorporated into the amplicons (see table below). Sequencing libraries are quantified using the NEBNext Library Quant Kit for Illumina (New England Biolabs). Subsequently, libraries from 20 samples are pooled in equimolar amounts.
  • Z-scores per chromosome arm are squared and then summed to derive a genome-wide aneuploidy score for each patient. Based on the cutoff established by Belie et aL, a genome-wide aneuploidy score is categorized as either high (> 5) or low ( ⁇ 5).
  • a PCR assay is then developed based on a cocktail mix used to amplify amplicons in the target regions simultaneously.
  • ROC Receiver Operating Characteristic
  • the PCR test focused on sensitivity is combined with a highly specific NGS (Next- Generation Sequencing) which is performed only in positive patients (occurring in less than 0.5% of samples).
  • NGS Next- Generation Sequencing
  • This method allows to achieve highly specific and sensitive results, while maintaining cost-effectiveness.
  • an initial PCR assay allows to establish the initial risk of lung cancer by using the biomarkers with highest sensitivity. Repetitive nasal samples are taken to further increase such values on early stage and screening patients.
  • Example 6 cfDNA in nasal sample of patients
  • cfDNA values ranging from 5 - 100 ng/pL at an average of 20 ng/pL from just 1 mL of nasal fluids. These values are at least 20 times higher than the ones obtained from 1 mL of plasma of lung cancer patients reported to be in the average of 0.838 ng/pL.
  • the amount of cfDNA required in the method of the invention is very low, meaning that all measurements can be done using at least one nasal sample from a subject with as little as 0.5 mL of a nasal fluid.
  • Example 7 Target genes and mutations with lung cancer patients Tag-sequencing analysis was performed by tagging the cfDNA/RNA from nasal fluid samples of lung cancer patients and healthy individuals. The panel used for this analysis can identify more than 150 lung cancer hotspots in 20 genes. These hotspots correspond to specific regions of the target genes that are associated with lung cancer.
  • the procedure of the sample preparation involves several steps including reverse transcription of cfRNA, PCR amplification of target sequences, purification of amplicons, size selection and NGS analysis as described in example 1 and 2.
  • 68 lung cancer hotspots are mutations found in the target genes ALK, EGFR, KRAS, MAP2K1 , MET, NRAS, PIK3CA and TP53. They have been found using nasal fluid samples from lung cancer patients.
  • anticancer agents such as Alectinib is used as a treatment of patients having mutations in ALK and Mobocertinib is used as a treatment of patients having EGFR mutation.
  • patients under treatment with a known mutation in the target genes of the invention can be monitored carrying out a PCR reaction according to the method of the invention using nasal fluid samples to follow patient over time for their response to treatment.
  • Example 8 Performance of the method of the invention with lung cancer patients Nasal samples from 7 lung cancer patients have been tested for the target genes ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1 , MET, NRAS, PIK3CA, ROS1 and TP53.
  • the method of the invention was performed using ddPCR and NGS as described in example 1 and 2.
  • sample 1 is positive for ALK, EGFR and TP53
  • sample 2 is positive for TP53
  • sample 3 is positive for ALK
  • EGFR sample 4 is positive for EGFR
  • TP53 sample 5 is negative for the target genes tested
  • sample 6 is positive for PIK3CA
  • sample 7 is positive for MAP2K1 and TP53.
  • the 3 control samples from healthy subjects are negative.
  • each lung cancer patient has a specific profile of the target genes selected from the group comprising ALK, EGFR, MAP2K1 , PIK3CA and TP53.
  • Example 9 Analysis of samples from patients with a KRAS mutation
  • Digital droplet PCR was performed using nasal fluids from 6 patients having lung cancer with a known KRAS mutation according to the protocol described in example 1 .
  • a VAF of 0.5 means that 0.5% of the analyzed molecules (cfDNA) contained that mutation indicating that there is ctDNA in the sample pool.
  • cfDNA the analyzed molecules contained that mutation indicating that there is ctDNA in the sample pool.
  • all nasal samples from lung cancer patients have a positive VAF indicating that all nasal samples contain ctDNA corresponding to the target gene KRAS.
  • Example 10 Nasal microbiome analysis of patients with lung cancer
  • V4 the fourth hypervariable domain (V4) of the microbial 16S ribosomal RNA (rRNA) gene has been amplified according to the method described in example 2.
  • the method of the present invention based on nasal samples is an easier way to screen the lung microbiome.
  • the lung microbiome has also been related with clinical factors such as smoking history and other lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis, asthma, idiopathic pulmonary fibrosis.
  • Example 1 1 Analysis of somatic copy number alterations of patients with lung cancer
  • Nucleic acid sequencing analysis has been performed to analyze somatic copy number alterations (SCNA) which are present in nasal samples from 6 lung cancer patients using Fast Aneuploidy Screening Test-Sequencing System (Fast-Seq) as described in example 5.
  • SCNA somatic copy number alterations
  • aneuploidy score obtained by using nasal samples comprising at least one or more cfDNA/ctDNA can be used as a primary screening step to evaluate tumor load without prior mutation knowledge.
  • the method is cost-effective and suitable for various metastatic contexts, relying on the common presence of aneuploidy in tumors and requiring only 1 nanogram of cfDNA.
  • This model includes the biomarkers as additional covariates alongside clinical and genetic factors.
  • the biomarkers incorporated into this model are described in tables 4 and 5. These biomarkers are considered potential risk factors for lung cancer.
  • the relative risk (RR) in this model would include these biomarkers alongside clinical and genetic variables.
  • the following risk factors are considered for lifestyle risk: Age (years), Gender, Race or Ethnic Group, Education, BMI (kg/m2), COPD, Emphysema, Personal history of cancer, Family history of lung cancer, Personal history of pneumonia, Smoking status (Current smoker, former smoker), Smoking duration (years), Smoking intensity (cigarettes per day), Pack-years of smoking, Years since cessation, Breathing upon exertion, Cough intensity, changes in voice, flu or pneumonia within the past 2 year, exposure to asbestos or other chemicals, current residence, Physical activity, Exposures (Coronary heart disease, Myocardial infarction, Stroke, Diabetes mellitus, Chronic kidney disease, Arthritis, Asthma), Personal history of cancer
  • RR exp(Pi * Smoking Status + (3 2 * Pack-years of Smoking + p 3 * Environmental Carcinogen Exposure + (3 4 * Family History of Lung Cancer + (3 5 * Weighted Genetic Risk Score + p 6 * Biomarker 1 + (3 7 * Biomarker 2 + p 8 * Biomarker 3 + ).
  • 3n e.g. Pi to (3s) are the coefficients or log odds ratios (log OR) estimated from the statistical modeling process. Variables such as smoking status and the biomarkers represent an individual's risk factor values and are included as covariates in the model.
  • 3n e.g. Pi to f3s are estimated during the modelbuilding process, through logistic regression. These coefficients represent the contribution of each variable to the overall risk of lung cancer when all other variables are held constant.

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Abstract

La présente invention concerne un procédé pour identifier si un sujet risque de développer un cancer du poumon, le procédé comprenant la détection dans au moins un échantillon nasal dudit sujet d'au moins un ou plusieurs ADN acellulaires (ADNcf) qui sont des fragments de gènes cibles choisis dans le groupe comprenant ALK, EGFR, MAP2K1, PIK3CA et TP53 ; et la détection de la présence ou de l'absence de variants génétiques dudit ou desdits ADNcf.
PCT/EP2024/083042 2023-11-21 2024-11-20 Procédé pour identifier si un sujet présente un risque de développer un cancer du poumon Pending WO2025109033A1 (fr)

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Citations (2)

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