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WO2019068761A1 - Méthodes d'identification de patients cancéreux présentant un risque élevé de développer des métastases - Google Patents

Méthodes d'identification de patients cancéreux présentant un risque élevé de développer des métastases Download PDF

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WO2019068761A1
WO2019068761A1 PCT/EP2018/076906 EP2018076906W WO2019068761A1 WO 2019068761 A1 WO2019068761 A1 WO 2019068761A1 EP 2018076906 W EP2018076906 W EP 2018076906W WO 2019068761 A1 WO2019068761 A1 WO 2019068761A1
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cancer
patient
exons
patients
metastasis
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Inventor
Dolores C. GARCIA OLMO
Susana OLMEDILLAS LOPEZ
Mariano Garcia Arranz
Damián GARCIA OLMO
Begoña AGUADO OREA
Ramón PEIRO PASTOR
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Institut De Recerca Biomedica De Lleida Fundacio Dr Pifarre
Consejo Superior de Investigaciones Cientificas CSIC
Universidad Autonoma de Madrid
Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz
Original Assignee
Institut De Recerca Biomedica De Lleida Fundacio Dr Pifarre
Consejo Superior de Investigaciones Cientificas CSIC
Universidad Autonoma de Madrid
Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz
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Publication of WO2019068761A1 publication Critical patent/WO2019068761A1/fr
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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
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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

Definitions

  • the present invention relates to the field of biomedicine and cancer. Specifically, it relates to an in vitro method of prognosis or for predicting the risk of suffering from metastasis in a patient having localized cancer disease based on the differential presence of exons in circulating cell free DNA (cfDNA) in cancer patients.
  • cfDNA circulating cell free DNA
  • metastases rather than primary tumors are responsible for most cancer deaths. To prevent these deaths, improved ways of diagnosis, prognosis and treatment of metastatic disease are needed.
  • Circulating cell-free DNA has emerged as a non-invasive alternative to conventional serial tissue biopsying for the analysis of tumor molecular features (Heitzer E. et al. Clin Chem. 2015, 61 (1 ), 1 12-23).
  • Increasing evidences support the potential clinical utility of this approach, known as liquid biopsy, in colorectal cancer (CRC), particularly at advanced stages (Toledo RA. et al. Oncotarget. 2016, 8(21 ), 35289-35300).
  • cfDNA-based surveillance is able to anticipate disease progression months ahead of standard imaging follow- up (Misale S et al. Nature, 2012, 486(7404), 532-6; Reinert T. et al. Gut. 2016, 65(4), 625-34).
  • cfDNA has been used in a very recent study to reflect tumor molecular dynamics in drug response of metastatic CRC patients, tracking the evolution of resistance mutations in KRAS pathway genes at different time points along treatment with anti-EGFR therapy (Toledo RA. et al. Oncotarget. 2016, 8(21 ), 35289-35300).
  • Circulating tumor DNA (ctDNA) fragments represent a minor proportion of the total cfDNA and, therefore, require extremely sensitive and specific detection techniques.
  • NGS next- generation sequencing
  • NGS of cfDNA has been recently applied to CRC in search of serial changes of mutational profiles and tumor load fluctuations for early detection of recurrence (Kim ST et al. Oncotarget. 2015, 6(37), 40360-9; Zhou J et al. PLoS One. 2016, 1 1 (7), e0159708; Sakai K et al. PLoS One. 2015, 10(5), e0121891 ; Tie J et al. Oncol. 2015, 26(8), 1715-22).
  • tumors shed an insufficient amount of DNA to be analyzed, especially, but not exclusively, at early stages of disease (Kim ST, Lee WS et al. Oncotarget.
  • MET amplification has been very recently detected by exome-sequencing in plasma of patients refractory to anti-EGFR therapy (Raghav K et al. Oncotarget. 2016, 7(34), 54627-54631 ).
  • exome-sequencing to track specific mutations is impaired by several factors.
  • One of the major hurdles is that the sensitivity of mutation detection is severely affected by the concentration of cfDNA in plasma, the background noise rate, the relative abundance of ctDNA and the capture efficiency (Klevebring D et al. PLoS One. 2014, 9(8), e104417).
  • These approaches usually require sequencing at a high depth, which considerably increases costs, and even at a very high read depth, mutations present at extremely low levels could still be undistinguishable from the sequencing background (Calvez-Kelm FL et al. Oncotarget. 2016, 8(1 1 ), 18166-18176).
  • the identified set of exons was successfully used to classify a small subset of patients that could not be initially classified attending to the selection criteria established in Table 2. Indeed, every member of the U group, namely those with locally advanced disease (pT4) or affected nodes (pN1 -N2) in the absence of distant metastasis, were correctly classified by the DPE algorithm as belonging to the M group.
  • the invention relates to an in vitro method for identifying exons which are differentially present in patients having metastasis (DPE) with respect to patients having localized cancer disease, wherein said method comprises the following steps:
  • cfDNA circulating cell-free DNA
  • N group circulating cell-free DNA
  • M group metastasized disease
  • the exome capturing for whole-exome sequencing is conducted by a method comprising hybridization of cfDNA in said blood, plasma or serum sample with probes substantially complementary to substantially all the coding DNA sequences in the patient's species genome.
  • the exon quantification is conducted by counting the number of sequence reads obtained by whole- exome sequencing that map to known exons when these are aligned to a reference genome sequence.
  • the quantification values of DPE have been normalized, preferably data normalization has been conducted by the trimmed mean of M values (TMM) method.
  • the second embodiment of the present invention refers to an in vitro method of prognosis or for predicting the risk of suffering from metastasis in a patient having localized cancer disease, the method comprising:
  • DPE metastatic patients
  • DPE are quantified by a method selected from the group consisting of next generation sequencing, quantitative PCR (qPCR), PCR- pyrosequencing, PCR-ELISA, DNA microarrays, branched DNA, dot-blot, Fluorescence In Situ Hybridization assay (FISH), and multiplex versions of said methods.
  • DPE are quantified by whole-exome sequencing using next generation sequencing.
  • said patient is a human patient.
  • said cancer is one characterized by presence of circulating tumor DNA (ctDNA) in the blood.
  • said cancer is colorectal cancer, preferably adenocarcinoma.
  • said differentially present exons are selected from the group consisting of the 379 exons found to be differentially present in colorectal cancer defined in Table 1 .
  • the third embodiment of the invention refers to an in vitro method for selecting a treatment for a patient having localized cancer disease wherein said method comprises selecting a treatment according to the classification of said patient according to its prognosis or risk of metastasis by a method as defined above.
  • the method further comprises storing the method results in a data carrier, preferably wherein said data carrier is a computer readable medium.
  • the fourth embodiment of the invention refers to a computer implemented method, wherein the method is as defined above.
  • the fifth embodiment of the present invention refers to the use of a kit for the prognosis or for predicting the risk of suffering from metastasis in a patient having localized cancer disease on the basis of the differential presence of exons in circulating cell-free DNA (cfDNA) according to a method as defined above, said kit comprising:
  • FIG. 1 Box Plot of DNA concentration in plasma of colorectal cancer patients. Metastatic patients (M) showed a higher median concentration of cell-free DNA (cfDNA) in plasma than non-metastatic ones (N). The distribution of cfDNA concentration in unclassifiable patients (U) shares common characteristics with both groups. (B) Size distribution of a cfDNA library from a patient showing a nucleosomal laddering pattern with fragment sizes of 302, 472 and 641 bp, including adapter sequences.
  • FIG. 1 Schematic workflow of the experimental procedure performed.
  • Cell-free DNA cfDNA
  • CRC colorectal cancer
  • FIG. 3 MA plots for selected Differentially Present Exons (DPE) (pv ⁇ 0.005).
  • the log ratio of fold-change (FC) is plotted on the y-axis, called M (from “minus") while in the x-axis the average of the normalized counts (Counts Per Million) are represented, called A values (from "average”).
  • a total number of 379 exons were obtained with EdgeR combining two different methods: (A) Likelihood Ratio Tests (LRT) with 297 exons and (B) Quasi-Likelihood F-tests (QLF) with 366 exons. Overpresent exons in metastatic (M) and non-metastatic (N) groups are represented with A and ⁇ , respectively.
  • FIG. 1 Bidimensional Principal Components Analysis (PCA) plot. Metastatic (M) and non- metastatic (N) patients are properly clustered and clearly separated. Unclassifiable patients (U) form another group between the limits of M and N, probably due to their intermediate characteristics. (!) High-risk patients subjected to prophylactic treatment (HIPEC, hyperthermic intraperitoneal chemotherapy); (#) Correctly predicted metastasis; (+) Exitus.
  • CUA Bidimensional Principal Components Analysis
  • cancer patient and “subject suffering from cancer” are used herein interchangeably. It may refer to those subjects diagnosed after a confirmatory test (e.g., biopsy and/or histology) and subjects suspected of having cancer.
  • a confirmatory test e.g., biopsy and/or histology
  • subject suspected of having cancer refers to a subject that presents one or more signs or symptoms indicative of a cancer and is being screened for cancer.
  • a subject suspected of having cancer encompasses for instance an individual who has received a preliminary diagnosis (e.g., an X-ray computed tomography scan showing a mass) but for whom a confirmatory test (e.g., biopsy and/or histology) has not been done or for whom the stage of cancer is not known.
  • the term further includes individuals in remission.
  • subject or “individual”' are used herein interchangeably to refer to all the animals classified as mammals and includes but is not limited to domestic and farm animals, primates and humans, for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents.
  • the subject is a male or female human being of any age or race.
  • terapéuticaally effective amount refers to an amount that is effective, upon single or multiple dose administration to a subject (such as a human patient) in the prophylactic or therapeutic treatment of a disease, disorder or pathological condition.
  • probe refers to synthetic or biologically produced nucleic acids, between 10 and 285 base pairs in length which contain specific nucleotide sequences that allow specific and preferential hybridization under predetermined conditions to target nucleic acid sequences, and optionally contain a moiety for detection or for enhancing assay performance.
  • a minimum of ten nucleotides is generally necessary in order to statistically obtain specificity and to form stable hybridization products, and a maximum of 285 nucleotides generally represents an upper limit for length in which reaction parameters can be easily adjusted to determine mismatched sequences and preferential hybridization.
  • Probes may optionally contain certain constituents that contribute to their proper or optimal functioning under certain assay conditions.
  • probes may be modified to improve their resistance to nuclease degradation (e.g., by end capping), to carry detection ligands (e.g., fluorescein), to carry ligands for purification or enrichment purposes (e.g. biotin) or to facilitate their capture onto a solid support (e.g., poly- deoxyadenosine "tails").
  • detection ligands e.g., fluorescein
  • biotin ligands for purification or enrichment purposes
  • solid support e.g., poly- deoxyadenosine "tails”
  • hybridization refers to a process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another.
  • substantially hybridization means that the amount of hybridization observed will be such that one observing the results would consider the result positive with respect to hybridization data in positive and negative controls. Data which is considered “background noise” is not substantial hybridization.
  • stringent hybridization conditions means approximately 35°C to 65°C in a salt solution of approximately 0.9 molar NaCI. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a rule, the stringency of the conditions under which hybridization is to take place will dictate certain characteristics of the preferred probes to be employed.
  • the invention relates to an in vitro method for identifying exons which are differentially present in patients having metastasis (DPE) with respect to patients having localized cancer disease, wherein said method comprises the following steps: i. quantifying individual exons by whole-exome sequencing of the circulating cell-free DNA (cfDNA), preferably by next-generation sequencing, in a biological fluid sample (e.g. blood, plasma or serum sample) obtained from a cancer patient or group of patients with localized disease (N group);
  • a biological fluid sample e.g. blood, plasma or serum sample
  • a biological fluid sample e.g. blood, plasma or serum sample obtained from a cancer patient or group of patients with metastasized disease (M group);
  • DPE refers to exons which are differentially present in patients having metastasis with respect to patients having localized cancer disease, i.e, exons which levels have been found to significantly differ between a patient group with localized disease with respect to a patient group with metastasis.
  • a method for identifying DPE has been defined under the first aspect of the invention.
  • exon refers to any part of a gene that will encode a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing.
  • exon may refer to the DNA sequence within a gene and/or to the corresponding sequence in RNA transcripts.
  • introns are removed and exons are covalently joined to one another as part of generating the mature messenger RNA.
  • the entire set of exons for a species constitutes the exome.
  • patient having localized cancer disease refers to a patient having a primary solid tumor in the absence of distant metastasis, preferably in the absence of regional lymph node metastasis or distant metastasis. For instance, it would correspond to tumors in stages TO, T1 , T2, T3 or T4 of the TNM system classification, preferably to stages TO, T1 , T2 or T3, with no signs of regional lymph node or distant metastasis (American Joint Committee on Cancer, AJCC. Chicago, Illinois. AJCC Cancer Staging Manual, 7th edition, published by Springer- Verlag New York, www.cancerstaging.org).
  • Metastasis refers to distant metastasis affecting organs other than the primary tumor site. Metastasis may be defined as the process by which cancer spreads or transfers from the primary site to other regions of the body with the development of a similar cancerous lesion at the new location (see for instance: Chambers AF et al., Nat Rev Cancer 2002; 2: 563-72), for instance in colorectal cancer metastasis in another organ (e.g., the liver) typically shows an enteroid adenocarcinoma pattern.
  • a "metastatic” or “metastasizing” cell is typically one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.
  • biological fluid sample includes biological fluids, such as whole blood, serum, plasma, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural effusion and urine.
  • biological fluid sample is blood, plasma or serum.
  • these types of samples are routinely used in the clinical practice and a person skilled in the art will know how to identify the most appropriate means for their obtaining and preservation.
  • Such biological samples can be taken around the time of diagnosis, before, during or after treatment (e.g. surgical resection).
  • the samples are obtained from patients diagnosed with cancer which have a resectable tumor (i.e., candidates for surgical resection).
  • the human genome contains approximately 3 billion base pairs (bp), 20.800 coding genes and over 200.000 exons, which represents about 1 -3% of the genome (Cunningham F et al. Nucleic Acids Res. 2015;43, D662-669; Clamp M, et al., Proc Natl Acad Sci USA. 2007; 104(49): 19428- 19433). On average, there are nine exons per gene, with an average exon size of 170 bp (Sakharkar MK et al., In Silico Biol. 2004, 4(4):387-393).
  • the term "whole-exome sequencing” as used herein refers to the sequencing of substantially all the coding genes in a genome.
  • exome capturing methods have been described and are well known in the art. These include polymerase chain reaction strategies, hybridization-based methods and the use of selective circularization probes which are described in further detail in Ballester et al (Expert Review of Molecular Diagnostics 2016, 16 (3), 1 -37) which is hereby incorporated by reference. Any of these exome-capturing methods can be used in the methods of the invention.
  • exome capture is based on a hybridization method comprising the use of sequences substantially complementary to the regions of interest (i.e. capture probes).
  • substantially complementary to the target sequence as used herein means that is at least about 90%, preferably at least about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence.
  • the capture probe comprises a sequence that is 100% complementary to a target polynucleotide sequence.
  • Capture-based methods for enrichment of coding genes may be solid-phase methods, typically using oligonucleotide microarrays or in-solution methods.
  • exome capturing for whole-exome sequencing is conducted by a method comprising hybridization of cfDNA in said blood, plasma or serum sample with probes specifically hybridizing to substantially all the coding DNA sequences in the subject's species genome.
  • substantially all coding sequences refers to at least about 90%, preferably about 95%, 96%, 97%, 98%, 99% or 100% of the coding sequences available in public databases.
  • probes may be modified with different ligands for target enrichment, for instance capture probes may be biotinylated.
  • these probes hybridize to the same sequences that the probes comprised in the SeqCap EZ probe pool provided by Roche NimbleGen to perform sequence capture.
  • the SeqCap EZ probe pool enriches for ⁇ 44 Mb of the human exonic regions.
  • the SeqCap system uses 55- to 105-base DNA biotinylated probes to capture known coding DNA sequences (CDS) from the NCBI Consensus CDS Database, RefSeq, and Sanger miRBase, for further details see "Whole-Exome Enrichment with the Roche NimbleGen SeqCap EZ Exome Library SR Platform" Chen et al., Cold Spring Harbor Protocols 2015.
  • exome capturing for whole-exome sequencing is conducted by a method comprising hybridization of cfDNA in said blood, plasma or serum sample with probes comprising or consisting of the probes in the SeqCap EZ probe pool, preferably, wherein these probes are biotinylated.
  • next-generation sequencing methods have been described and are well known to a person skilled in the art. These include for instance sequencing by synthesis with cyclic reversible termination approaches (e.g., Illumina, SEQLL, Qiagen), sequencing by synthesis with single-nucleotide addition approaches (e.g., Roche-454, Thermo Fisher-Ion Torrent), sequencing by ligation (e.g., Thermo Fisher SOLiD and BGI-Complete Genomics), real-time long-read sequencing (e.g., Pacific Biosciences, Oxford Nanopore Technologies), synthetic long-read sequencing (e.g., Illumina, 10X Genomics, iGenomeX), see for instance Goodwin S, et al., Nat Rev Genet. 2016, 17(6):333-51 ).
  • whole-exome sequencing is conducted by paired-end sequencing.
  • the depth of sequencing coverage when performing whole-exome sequencing for use in a method of the invention will typically be below the one used in applications aiming at identifying single nucleotide polymorphisms (SNPs), which have generally a read depth of about 100x.
  • whole-exome sequencing is characterized by an average read depth of 40-80x per sample.
  • Quantifying or “determining the levels ", as used herein, refers to ascertaining the absolute or relative amount or concentration of the exons in the sample. Techniques to assay levels of individual exons from test samples are well known to the skilled technician, and the invention is not limited by the means by which the components are assessed.
  • exon quantification is typically conducted by counting the number of sequence reads that map to known exons when these are aligned to a reference genome sequence.
  • the reference genome sequence will be of the same species as the subject from which a cfDNA sample has been obtained and an exome library has been prepared.
  • said subject is a human subject and said reference genome sequence is hg38.
  • DPE DPE those exons which are differentially over-present in the N group, those which are differentially over-present in the M group or those which are differentially over-present in either the N group or the M group; preferably, are classified as DPE those exons which are differentially over-present in either the N group or the M group.
  • in step iii) are classified as DPE those exons having a differential expression between the N and M group of more than about 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 fold levels deviation (i.e., increase or decrease).
  • a statistically significant difference between the target exon levels can be established by a person skilled in the art by means of using different statistical tools; illustrative, non-limiting examples of said statistical tools include determining confidence intervals, determining the p-value, the Chi-Square test discriminating functions, etc.
  • Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, at least 99%.
  • the p-values are, preferably less than 0.1 , less than 0.05, less than 0.01 , less than 0.005 or less than 0.0001.
  • exon quantification in the method of the invention is typically conducted by counting the number of sequence reads that map to known exons when these are aligned to a reference genome sequence.
  • Analysis of differential presence as described herein may be performed using edgeR software package for the differential expression of RNA-seq data as described herein.
  • RNA-seq data is typically summarized by counting the number of sequence reads that map to genomic features of interest.
  • Negative binomial models are used in the edgeR software to capture the quadratic mean-variance relationship that can be observed in the RNA-seq data.
  • Empirical Bayes methods are used to allow exon-specific variation estimates.
  • differentially expressed exons are identified using either or both of the Likelihood Ratio Tests (LRT) Quasi-Likelihood F-tests (QLF) as statistical methods.
  • quantification values of DPE have been normalized, preferably data normalization has been conducted by the trimmed mean of M values (TMM) method described in Robinson MD et al. (Bioinformatics. 2010, 26(1 ), 139-40) and used by the EdgeR software.
  • TMM trimmed mean of M values
  • the present invention provides an in vitro method of prognosis or for predicting the risk of suffering from metastasis in a patient having localized cancer disease, the method comprising:
  • DPE blood, plasma or serum sample obtained from said patient
  • ii comparing the quantification values in the patient's sample obtained in i) with the quantification values in a reference sample, wherein said reference sample is isolated from a patient or group of patients suffering from localized cancer disease; wherein when the quantification values in the patient's sample are increased or decreased in comparison with those in the reference sample the patient has a high risk of suffering from metastasis.
  • reference value relates to a predetermined criteria used as a reference for evaluating the values or data obtained from the samples collected from a subject.
  • the reference value or reference level can be an absolute value, a relative value, a value that has an upper or a lower limit, a range of values, an average value, a median value, a mean value, or a value as compared to a particular control or baseline value.
  • a reference value can be based on an individual sample value or can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested.
  • the reference value according to the method of the invention can be obtained from one or more subjects having localized cancer disease, from subjects suffering from metastasis, from subjects suffering from cancer at early stage, such as non-symptomatic (preclinical stage) or from the same subject that was diagnosed as having cancer but at an earlier time point.
  • said reference sample is isolated from a patient or group of patients suffering from localized cancer disease, and when the quantification values of said set of individual exons identified as differentially present in metastatic patients (DPE) are significantly different from those in the reference sample the patient has a high risk of suffering from metastasis.
  • DPE metastatic patients
  • said reference sample is isolated from a patient or group of patients suffering from metastasis; and when the quantification values of said set of individual exons identified as differentially present in metastatic patients (DPE) in the patient's sample are significantly different from those in the reference sample, the patient has a low risk of suffering from metastasis.
  • DPE metastatic patients
  • the method of the invention is a method of monitoring cancer progression and the reference value is obtained from the same subject that was diagnosed as having cancer but at an earlier time point.
  • the level of presence (i.e. abundance) of an exon is considered "decreased" when the level of said exon in a sample is lower than its reference value.
  • the level is considered to be lower than its reference value when it is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 140%, at least 150%, or more lower than its reference value; more preferably, the level is considered to be lower than its reference value when it is at least 20% lower than its reference value.
  • the level of presence (i.e. abundance) of an exon is considered “increased” when the level of said exon in a sample is higher than its reference value.
  • the level is considered to be higher than its reference value when it is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 140%, at least 150%, or more higher than its reference value; more preferably the level is considered to be higher than its reference value when it is at least 20% higher than its reference value.
  • subjects having more than about 1 .1 ,1 .2, 1.3, 1 .4, 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 fold levels deviation (i.e., increase or decrease) than the reference value as described herein, preferably having more than about 1 .2 folds levels deviation than the reference value, may be identified as being differentially present.
  • Metastasis prediction in the method of the invention does not claim to be correct in 100% of the analyzed samples. However, it requires that a statistically significant amount of the analyzed samples are classified correctly.
  • the amount that is statistically significant can be established by a person skilled in the art by means of using different statistical tools; illustrative, non-limiting examples of said statistical tools include determining confidence intervals, determining the p-value, the Chi-Square test discriminating functions, etc.
  • Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, at least 99%.
  • the p-values are, preferably less than 0.1 , less than 0.05, less than 0.01 , less than 0.005 or less than 0.0001.
  • the teachings of the present invention preferably allow correctly diagnosing in at least 60%, in at least 70%, in at least 80%, or in at least 90% of the subjects of a determining group or population analyzed.
  • solid tumors There are different types of solid tumors which are typically named according to the type of cells that form them.
  • solid tumors are lung cancer, sarcoma, malignant melanoma, mesothelioma, bladder carcinoma, prostate cancer, pancreas carcinoma, gastric carcinoma, ovarian cancer, hepatoma, breast cancer, colorectal cancer, kidney cancer, esophageal cancer, suprarenal cancer, parotid gland cancer, head and neck carcinoma, cervix cancer, mesothelioma and lymphoma.
  • the differentially present exons detected using the method of the invention can probably come from tumor and non-tumor derived cfDNA.
  • the localized cancer disease is one characterized by presence of circulating tumor DNA (ctDNA) in the blood, in particular where the amount of ctDNA arising from the primary solid tumor is such that exons can be quantified from a blood, plasma or serum sample by molecular methods known in the art such as next-generation sequencing.
  • This method being particularly useful for the determination of metastasis risk in those cancer types where one of the mechanisms involved in metastasis mediation is the dissemination of acellular material in the plasma (e.g. exosomes, microparticles, lysosomes).
  • cancer types where presence of circulating tumor DNA has been described include colorectal cancer, breast cancer, lung cancer, ovarian cancer and pancreatic cancer (Nature Reviews Cancer
  • said solid tumor is a carcinoma.
  • carcinoma refers to a malignant neoplasm of epithelial origin or cancer of the internal or external lining of the body. Carcinomas are divided into two major subtypes: adenocarcinoma, which develops in an organ or gland, and squamous cell carcinoma, which originates in the squamous epithelium.
  • Carcinoma can be classified according to the site of origin or cell type and include inter alia: breast carcinoma; ovarian carcinoma; cervix carcinoma; gastric carcinoma; non-small cell lung carcinoma; small cell lung carcinoma; pancreatic carcinoma; prostate carcinoma; colon carcinoma; liver carcinoma; renal carcinoma; bladder carcinoma; prostate carcinoma; head and neck carcinoma; squamous cell carcinoma; epidermoid carcinoma; choriocarcinoma; seminoma; embryonal cell carcinoma; and mesothelioma.
  • said carcinoma is colon adenocarcinoma.
  • said cancer is colorectal cancer.
  • said cancer is colorectal cancer and said differentially present exons are selected from the group consisting of the 379 exons found to be differentially present in colorectal cancer defined in Table 1 below.
  • Table 1 DPE between CRC patients with metastatic and localized disease (EnsembI Exon ID; www.ensembl.org/); Version: EnsembI GRCh38.p2 (dec 2014).
  • ENSE00000924669 ENSE00001068195 ENSEOOOO 1245555 ENSE00000926037 ENSE00001072073 ENSEOOOO 1252408
  • ENSEOOOO 1327628 ENSEOOOO 1500245 ENSEOOOO 1677669
  • the accuracy of the method of the invention can be increased by determining the presence and/or the quantification of other biomarkers which have been described to be associated to metastasis (see for instance, Martini G, Troiani T, Cardone C,et al., World J Gastroenterol. 2017 23(26):4675-4688) and/or clinical signs or symptoms with reported prognostic/predictive value, such as morphological features of the tumor, histological subtypes, radiological traits of the imaging tests (e.g. size, shape, volume, radiological texture, morphological details or other features in a CT scan, X-Ray or SUV or alternative ways to analyze nuclear tracer levels in a PET imaging, etc); clinical characteristics of the patients (e.g.
  • the potential additional biomarkers to be associated to the present invention can be found in the tumor specimen itself or other cells, or other bodily sample, such as body fluids (e.g. blood or plasma), feces or exhaled breath obtained from the same patient.
  • body fluids e.g. blood or plasma
  • feces or exhaled breath obtained from the same patient.
  • the methods of the invention further comprise determining the presence and/or the quantification of other biomarkers, clinical signs and/or symptoms, and/or clinical characteristics of the patients predictive of metastasis.
  • biomarker refers to markers of disease which are typically substances found in a bodily sample which generally can be easily measured. Typically, the measured amount correlates to an underlying disease pathophysiology, making it useful for diagnosing, predicting and/or measuring the progress of a disease or the effects of a treatment.
  • biomarker encompasses biophysical and biochemical determinations, including genetic and serological markers.
  • the method of prognosis or for predicting the risk of suffering from metastasis in a cancer patient of the invention may be used for the classification or selection of patients as belonging to a particular group of risk (i.e. those patients with an increased risk of suffering from metastasis).
  • a treatment may be selected or personalized according to the risk group into which the cancer patient has been classified. For instance, patients in the high risk group will be treated with the best possible treatment (i.e, surgical, adjuvant and/or neoadjuvant treatment) and treatment regimen; whereas a less aggressive treatment or no treatment will be selected for those patients with lower risk of suffering from metastasis.
  • the invention relates to an in vitro method for selecting the treatment for a patient having localized cancer disease, wherein said method comprises determining the prognosis or predicting the risk of suffering from metastasis in said cancer patient by a method as defined in previous aspects of the invention.
  • the invention refers to a method for the treatment of a patient having localized cancer disease comprising administering to said patient a therapeutically effective amount of a treatment (e.g. a drug or drug combination) wherein said treatment is selected according to the classification of said cancer patient according to its risk of suffering from metastasis, wherein said risk has been determined by the method of prognosis or for predicting the risk of suffering from metastasis according to the invention.
  • a treatment e.g. a drug or drug combination
  • This treatment may be a neoadjuvant treatment administered prior to the surgical removal of the tumor and/or an adjuvant treatment after the surgical intervention.
  • the invention refers to an in vitro method of monitoring disease progression or response to a treatment in a patient having localized cancer disease, wherein said method comprises determining the prognosis or the risk of suffering from metastasis in said cancer patient by a method according to the invention.
  • monitoring refers to determining the evolution of the disease and/or the efficacy of a therapy, for example determining whether there has been a change in the levels of a set of DPE which is indicative of worst prognosis or increased risk of metastasis.
  • One of the goals of the method of monitoring of the invention is to early detect an increased risk of metastasis. Preferred embodiments and features of the invention are as described under previous aspects.
  • a further aspect of the invention refers to a computer implemented method, wherein the method is any of the methods disclosed herein or any combination thereof.
  • any computer program capable of implementing any of the methods of the present invention or used to implement any of these methods or any combination thereof also forms part of the present invention.
  • any device or apparatus comprising means for carrying out the steps of any of the methods of the present invention or any combination thereof, or carrying a computer program capable of, or for implementing any of the methods of the present invention or any combination thereof, is included as forming part of the present specification.
  • the methods of the invention may also comprise the storing of the method results in a data carrier, preferably wherein said data carrier is a computer readable medium.
  • the present invention further relates to a computer-readable storage medium having stored thereon a computer program of the invention or the results of any of the methods of the invention.
  • a computer readable medium can be any apparatus that may include, store, communicate, propagate, or transport the results of the determination of the method of the invention.
  • the medium can be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (or apparatus or device) or a propagation medium.
  • the invention further relates to a kit as defined herein below and to the use of said kit for the prognosis or for predicting the risk of suffering from metastasis in a patient having localized cancer disease on the basis of the differential presence of exons in circulating cell-free DNA (cfDNA) according to a method of the invention, said kit comprising:
  • a reagent for quantifying individual exons in a biological fluid sample e.g. blood, plasma or serum sample
  • nucleic acids quantification are well known in the art and have been described herein above. This may include next generation sequencing, quantitative PCR (qPCR), PCR- pyrosequencing, PCR-ELISA, DNA microarrays, branched DNA, dot-blot, Fluorescence In Situ Hybridization assay (FISH), and multiplex versions of said methods.
  • qPCR quantitative PCR
  • PCR-pyrosequencing PCR-pyrosequencing
  • PCR-ELISA DNA microarrays
  • DNA microarrays branched DNA
  • dot-blot dot-blot
  • FISH Fluorescence In Situ Hybridization assay
  • the kit of the invention will therefore include reagents according to the selected method for exon quantification.
  • said kit comprises reagents suitable for performing a real-time or qPCR reaction, which typically contain a DNA polymerase, such as Taq DNA polymerase (e.g., hot- start Taq DNA polymerase), buffer, magnesium, dNTPs, and optionally other agents (e.g., stabilizing agents such as gelatin and bovine serum albumin).
  • a DNA polymerase such as Taq DNA polymerase (e.g., hot- start Taq DNA polymerase)
  • buffer such as buffer, magnesium, dNTPs, and optionally other agents (e.g., stabilizing agents such as gelatin and bovine serum albumin).
  • agents e.g., stabilizing agents such as gelatin and bovine serum albumin.
  • real-time PCR reaction mixtures also contain reagents for real time detection and quantification of amplification products.
  • said kit comprises reagents suitable for whole-exome sequencing of cfDNA by next generation sequencing, for instance it will comprise reagents suitable for whole-exome capturing and/or for next generation sequencing.
  • kit may comprise for instance reagents typically used in DNA extraction protocols (e.g. wash and/or dilution buffers, proteinase, etc.) and/or reagents usually used in the capturing of nucleic acid sequences (e.g. buffers, magnesium, probes, etc.).
  • said patient has colorectal cancer and said kit comprises reagents suitable for the quantification of individual exons selected from the group consisting of the 379 exons found to be differentially present in colorectal cancer defined in Table 1 .
  • An in vitro method for identifying exons which are differentially present in patients having metastasis (DPE) with respect to patients having localized cancer disease comprising the following steps:
  • exome capturing for whole-exome sequencing is conducted by a method comprising hybridization of cfDNA in said blood, plasma or serum sample with probes substantially complementary to substantially all the coding DNA sequences in the patient's species genome.
  • exon quantification is conducted by counting the number of sequence reads obtained by whole-exome sequencing that map to known exons when these are aligned to a reference genome sequence.
  • step iii) are classified as DPE those exons which quantification levels differ between the two groups in at least 1.2- fold.
  • step iii) Likelihood Ratio Tests (LRT) and/or Quasi-Likelihood F-tests (QLF) are used as statistical methods for identifying differentially present exons.
  • LRT Likelihood Ratio Tests
  • QLF Quasi-Likelihood F-tests
  • TMM trimmed mean of M values
  • DPE metastatic patients
  • DPE are quantified by a method selected from the group consisting of next generation sequencing, quantitative PCR (qPCR), PCR- pyrosequencing, PCR-ELISA, DNA microarrays, branched DNA, dot-blot, Fluorescence In Situ Hybridization assay (FISH), and multiplex versions of said methods.
  • DPE are quantified by whole-exome sequencing using next generation sequencing.
  • the method according to any of the precedent items wherein said patient is a human patient.
  • the method according to any of the precedent items, wherein said cancer is one characterized by presence of circulating tumor DNA (ctDNA) in the blood.
  • the method according to any of the precedent items, wherein said cancer is selected from the group consisting of colorectal cancer, breast cancer, lung cancer, ovarian cancer and pancreatic cancer.
  • said cancer is colorectal cancer, preferably adenocarcinoma.
  • said differentially present exons are selected from the group consisting of the 379 exons found to be differentially present in colorectal cancer defined in Table 1.
  • An in vitro method for selecting a treatment for a patient having localized cancer disease wherein said method comprises selecting a treatment according to the classification of said patient according to its prognosis or risk of metastasis by a method as defined in any of items 8 to 15.
  • An in vitro method of monitoring disease progression or response to a treatment in a patient having localized cancer disease comprises determining the prognosis or the risk of suffering from metastasis in said cancer patient by a method as defined in any of items 8 to 15.
  • said method further comprises storing the method results in a data carrier, preferably wherein said data carrier is a computer readable medium.
  • kit 21 Use of a kit according to item 20, wherein said kit comprises reagents suitable for whole- exome capturing and/or for next generation sequencing.
  • kits according to any of items 20 or 21 , wherein said patient has colorectal cancer and said kit comprises reagents suitable for the quantification of individual exons selected from the group consisting of the 379 exons found to be differentially present in colorectal cancer defined in Table 1.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • the term “comprises” also encompasses and expressly discloses the terms “consists of” and “consists essentially of”.
  • the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation.
  • words of approximation such as, without limitation, "about”, “around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present.
  • the extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as "about” may vary from the stated value by ⁇ 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10%.
  • the term "about” may mean the indicated value ⁇ 5% of its value, preferably the indicated value ⁇ 2% of its value, most preferably the term “about” means exactly the indicated value ( ⁇ 0%).
  • the following examples serve to illustrate the present invention and should not be construed as limiting the scope thereof.
  • Plasma samples were collected before surgery in EDTA tubes and centrifuged at 1800 x g for 10 minutes. Plasma obtained from the first centrifugation was centrifuged again at 3000 x g for 10 min, aliquoted and stored at -80°C until analysis.
  • Circulating cell-free DNA was extracted from plasma with the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Concentration, quantity and integrity of cfDNA were estimated prior to use. The size distribution of the cfDNA fragments was determined using an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). Additional shearing was not performed since the majority of circulating DNA fragments in plasma is naturally short. Library preparation and specific exome capture were performed using the SeqCap EZ HGSC VCRome Kit (Roche NimbleGen, Basel, Switzerland). This protocol is based on the Roche NimbleGen SeqCap EZ Exome Library SR platform, for further details see Chen et al.
  • cfDNA sequencing data were processed as in a typical RNA-seq pipeline, but this strategy was aimed at detecting presence of exons instead of gene expression.
  • DGE digital gene expression
  • NGS Next Generation Sequencing
  • glm generalized linear models
  • the glm functions can test for differential expression using either likelihood ratio tests (LRT) (Robinson MD et al. Bioinformatics. 2010, 26(1 ), 139-40) or quasi- likelihood F-tests (QLF) (Lund SP et al. Stat Appl Genet Mol Biol. 2012;1 1 (5)).
  • LRT likelihood ratio tests
  • QLF quasi- likelihood F-tests
  • EdgeR uses the trimmed mean of M values (TMM) for data normalization (Robinson MD et al. Bioinformatics. 2010, 26(1 ), 139-40; McCarthy, J. D, et al., Nucleic Acids Research 2012, 40(10), 4288-4297).
  • TMM trimmed mean of M values
  • PCA Principal Components Analysis
  • RF classification was implemented with an R script using YandomForest' package (Liaw A. R News. 2002, 2(3), 5). Briefly, 2 samples from M and N were randomly selected and extracted from each group, respectively, using the 8 remaining samples (16 samples in total) as a 'training set' to generate a predictive algorithm. One hundred classifications were performed by iteration of this process and the mean value of the obtained probabilities was calculated. The accuracy of the resulting model was tested by checking its ability to correctly classify previously extracted samples into their corresponding groups of origin.
  • Circulating cell-free DNA was successfully extracted from all plasma samples, obtaining a variable concentration of DNA that ranged from 13.76 to 1602.90 pg/ ⁇ -. Median DNA concentration was higher in metastatic patients in comparison to non-metastatic patients (Fig. 1A), although this difference was not statistically significant. Unclassifiable patients' DNA median concentration was slightly elevated with respect to both groups. Bioanalyzer plots revealed a characteristic cfDNA sizing distribution with a nucleosomal fragmentation pattern. We obtained cfDNA with median fragment lengths of 173 and 342 bp, once adapter sequencing lengths (126 nt) were substracted. One additional peak of 51 1 bp was only observed in 2 patients of our series (Fig. 1 B and Table 3).
  • the total number of reads per patient ranged from 45 to 87 million reads with a read length of 76 bp (see Table 3 for further details).
  • Quality analyses performed over reads using FastQC software indicated that base calling quality (Phred+33 quality score) was maintained in general good standard across all cycles with median and mean base quality over 28, although some bases' quality fell down to 22. As usual, a certain lack of accuracy was found in 10-1 1 first bases.
  • the DPE was analyzed with EdgeR using either likelihood ratio tests (LRT) or quasi-likelihood F-tests (QLF) tests, with a threshold of p-value ⁇ 0.005 for M ⁇ N comparison.
  • LRT likelihood ratio tests
  • QLF quasi-likelihood F-tests
  • MA plots for selected DPE are represented in Figure 3, wherein are highlighted those exons which are differentially over-present (i.e. wherein the exon levels are significantly increased) in the N group or differentially over-present in the M group.
  • Clusterization of normalized quantification values of the 379 identified DPE was performed by Ward's method. The resulting tree is included in Figure 4. As observed, patients are mostly grouped properly, keeping the M and the N samples separated.
  • FIG. 5 represents a bidimensional plot with the two first Principal Components.
  • M and N groups are clearly separated and cluster properly.
  • U patients are located between the limits of both groups, supporting the idea that patients belonging to the U group share characteristics with metastatic as well as non-metastatic patients.
  • RF Random Forest
  • a checking test was performed to confirm whether the algorithm was able to classify extracted samples into their corresponding groups of origin, calculating the average probabilities of belonging to one group or another (Table 3). Thus, extracted samples were correctly identified, with the highest mean probability being 0.68.

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Abstract

L'invention concerne des méthodes d'identification de patients cancéreux présentant un risque élevé de développer des métastases. La présente invention concerne le domaine de la biomédecine et du cancer. Spécifiquement, l'invention concerne une méthode in vitro de pronostic ou de prédiction du risque de souffrir de métastases chez un patient ayant une maladie cancéreuse localisée sur la base de la présence différentielle d'exons dans l'ADN acellulaire circulant (cfDNA) chez des patients cancéreux.
PCT/EP2018/076906 2017-10-03 2018-10-03 Méthodes d'identification de patients cancéreux présentant un risque élevé de développer des métastases Ceased WO2019068761A1 (fr)

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