Patent Application
20180363057
The present invention relates to the field of the medicine. In particular, it relates to methods for evaluating the radio-sensitivity and the risk of adverse effects for an individual.
Inter-individual differences in radio-sensitivity are extensively documented since the discovery of radiation, and are causally related to the varying levels of toxicity that all radiotherapy patients will experience. However, except for very rare monogenic diseases such as homozygous carriers of a mutated ATM gene, the genetic basis of individual radio-sensitivity is poorly characterized. Nevertheless, numerous studies have indicated a correlation between cellular- and clinical radio-sensitivity. This is exemplified by the association between low CD8+-lymphocyte apoptosis and radiation induced late toxicity (Ozsahin et al., 2005, Clin Cancer Res. 11:7426-7433). However, no consensus exists on available biological tests that can be reliably used for prediction of early- and/or late clinical adverse effects associated with radiotherapy (Finnon et al., 2012, Radiother Oncol. 105:329-336; Greve et al., 2011, PLoS ONE. 7:e47185- e47185).
Dose limitation due to side-effects observed in a minority of patients greatly reduces efficacy of radiotherapy, because of the direct relationship between radiation dose and tumor control. Beyond technical developments such as optimizing radiation delivery, improving radiotherapy outcome requires a better understanding of the underlying mechanisms of individual radio sensitivity (West and Barnett, 2011, Genome Med. 3:52) that will ultimately allow individualized radiotherapy.
Therefore, methods allowing to foreseeing radio sensitivity would be useful for the clinicians in order to optimize the cancer therapy.
The present invention provides a method for assessing the individual radio sensitivity associated to apoptosis susceptibility of lymphocytes and risk of developing complications in irradiated normal tissues. The method comprises genotyping particular polymorphisms in TNFSF10/TRAIL gene.
The present invention relates to the use of a single nucleotide polymorphism (SNP) selected from the group consisting of rs1131532, rs3815496 and any SNP having a linkage disequilibrium with an absolute value for r2 of 0.85 therewith as a biomarker for assessing the risk of developing adverse effects after radiotherapy in a subject, wherein said adverse effects are selected for the group consisting of acute and/or subacute dermatitis and lymphocyte apoptosis.
The present invention also relates to a method for assessing the risk of developing adverse effects after radiotherapy in a subject, wherein the method comprises determining the allele of a single nucleotide polymorphism (SNP) selected from the group consisting of rs1131532, rs3815496 and any SNP having a linkage disequilibrium with an absolute value for r2 of 0.85 therewith in a subject sample; wherein said adverse effects are acute and/or subacute dermatitis and/or lymphocyte apoptosis. Preferably, the step of determining the allele is a step of genotyping by sequencing, selective hybridization and/or selective amplification, enzyme-based methods or methods relying on differences in the conformation, weight or size of the molecules. Preferably, the subject sample is saliva, urine, whole blood, plasma, or serum sample. More preferably the subject is human.
The present invention also relates to a method for determining the radiation dose suitable for a subject, comprising performing the method for assessing the risk of developing adverse effects after radiotherapy in a subject as disclosed above, and selecting the suitable radiation dose for the subject so as a reduced radiation dose is selected if the subject has at least one allele of the SNP associated with an increased risk of developing adverse effects after radiotherapy and a maximal radiation dose is selected if the subject does not present any allele of the SNP associated with an increased risk of developing adverse effects after radiotherapy.
The present invention further relates to the use of a kit for assessing the risk of developing adverse effects after radiotherapy in a subject, said adverse effects being acute and/or subacute dermatitis and/or lymphocyte apoptosis, or for determining the radiation dose and/or selecting the radiotherapy delivery suitable for a subject, wherein the kit comprises at least one nucleic acid probe capable of specifically binding or hybridizing a SNP according to the invention, and/or at least one set of primers suitable for use in amplification reaction suitable for amplifying a SNP as defined in the present invention.
Preferably, the SNP having a linkage disequilibrium with r53815496 is selected in one of the following groups consisting of
Alternatively, the SNP having a linkage disequilibrium with rs1131532 is selected in one of the following groups consisting of
In a preferred embodiment, the SNP having a linkage disequilibrium with rs1131532 and rs3815496 is selected from the group consisting of rs9811673, rs9811639, rs3815496, rs3181143, rs3136606, rs3136607, rs3819773, rs1126161, rs63562861, rs1131542, rs11720451, rs9859809, rs6807069, rs6783667, rs9869255, rs9811650, rs9811646, rs62282578, rs1992868, rs1992869, rs6414542, rs1597086, rs9880799 and rs4894557.
In a most preferred embodiment, the SNP is rs3815496 or rs1131532.
The inventors identify SNPs located in the TRAIL/TNFSF10 gene which can be used for predicting the radio-sensitivity of a subject. In particular, the identified SNP are associated with higher risk or probability of acute and/or subacute dermatitis, and/or lymphocyte apoptosis. The identified SNPs are rs53815496 and rs1131532 which present a linkage disequilibrium between each other.
In a first aspect, the identified SNPs and any SNP having a linkage disequilibrium with an absolute value for r2 of at least 0.85 can be used to determine radio-sensitivity of a subject after a radiation exposure, especially an accidental radiation exposure for instance due to ingestion, inhalation or deposition of radioactive materials. Then, if the subject is determined to present radio-sensitivity, he can benefit of an increased medical monitoring or receive an appropriate treatment such as preventive treatment.
However, the main source of radiation exposure is the radiotherapy, in particular for cancer treatment. Therefore, the main aspect of the present invention is the use of the identified SNPs and any SNP having a linkage disequilibrium with an absolute value for r2 of at least 0.85 as a biomarker for assessing the risk of developing adverse effects after radiotherapy in a subject. In particular, the adverse effects are selected for the group consisting of acute and/or subacute dermatitis and lymphocyte apoptosis, especially of CD4+ effector memory lymphocytes.
The term “radiotherapy” is commonly used in the art to refer to multiple types of radiation therapy including internal and external radiation therapy, radioimmunotherapy, and the use of various types of radiation including X-rays, gamma rays, alpha particles, beta particles, photons, electrons, neutrons, radioisotopes, and other forms of ionizing radiation. Preferably, the radiotherapy involves the use of X-rays or gamma-rays.
The term “TRAIL/TNFSF10 gene” as used herein, refers to a polynucleotide encoding a Tumor necrosis factor (ligand) superfamily, member 10 polypeptide (Gene ID: 8743). An exemplary sequence for this protein is Genbank Accession Number: NP_003801.1. An exemplary sequence for the polynucleotide (mRNA) encoding this precursor is Genbank Accession Number: NM_003810.3.
The methods of the invention as disclosed herein may be in vivo, ex vivo or in vitro methods, preferably in vitro methods.
The present invention further relates to a method for assessing the risk of developing adverse effects after radiotherapy in a subject, wherein the method comprises determining the allele of a single nucleotide polymorphism (SNP) selected from the group consisting of rs1131532, rs3815496 and any SNP having a linkage disequilibrium with an absolute value for r2 of 0.85 therewith in a subject sample. Preferably, said adverse effects are acute and/or subacute dermatitis, and/or lymphocyte apoptosis.
In a particular aspect, the presence of at least one allele A of SNP rs3815496 is indicative of a risk of developing adverse effects after radiotherapy or a predisposition to adverse effects after radiotherapy. The a risk of developing adverse effects after radiotherapy is higher in a subject carrying one or two alleles A of SNP rs3815496 compared to a subject only carrying alleles G. The risk or predisposition is increased in a subject who is a homozygous carrier of the allele A in comparison to a heterozygous carrier.
In another particular aspect, the presence of at least one allele C of SNP rs1131532 is indicative of a risk of developing adverse effects after radiotherapy or a predisposition to adverse effects after radiotherapy. The a risk of developing adverse effects after radiotherapy is higher in a subject carrying one or two alleles C of SNP rs1131532 compared to a subject only carrying alleles G. The risk or predisposition is increased in a subject who is a homozygous carrier of the allele C in comparison to a heterozygous carrier.
The present invention further relates to a method for optimizing the treatment to a subject, the method comprising performing the method for assessing the risk of developing adverse effects after radiotherapy in a subject as detailed above.
If the subject has at least one allele of the SNP associated with an increased risk of developing adverse effects after radiotherapy, he can benefit of an increased medical monitoring or receive an appropriate treatment such as preventive treatment. For instance, the preventive treatment can include a treatment with recombinant TRAIL or TRAIL agonistic antibodies. This treatment could be provided to the subject before the radiotherapy.
Depending on the risk of developing adverse effects after radiotherapy for a subject, the physician can optimize the radiotherapy procedure to the subject, for instance by selecting the most appropriate delivery means and/or selecting the radiation dose.
In a particular embodiment, the method is suitable for determining the radiation dose suitable for a subject. The method comprises performing the method for assessing the risk of developing adverse effects after radiotherapy in a subject as detailed above and selecting the suitable radiation dose for the subject so as a reduced radiation dose is selected if the subject has at least one allele of the SNP associated with an increased risk of developing adverse effects after radiotherapy and a maximal radiation dose is selected if the subject does not present any allele of the SNP associated with an increased risk of developing adverse effects after radiotherapy. The method may further comprises a step of administering/applying the selected radiation dose to the subject. In a preferred embodiment, the selected radiation dose is administered by conformational irradiation. Indeed, this technology is suitable for the delivery of a precise radiation dose to the subject at a precise location.
The inventors identified two SNPs, namely rs3815496 and rs1131532.
with R being A or G.
with B being C, G or T.
rs3815496 and rs1131532 present a linkage disequilibrium with an absolute value for r2 of 0.895 in population European super population and 1 in African super population.
A “polymorphic site” or “polymorphism site” or “polymorphism” or “single nucleotide polymorphism site” (SNP site) or “single nucleotide polymorphism” (SNP) as used herein is the locus or position with a given sequence at which divergence occurs. A “polymorphism” is the occurrence of two or more forms of a gene or position within a gene (allele), in a population. Polymorphic sites have at least two alleles, each occurring at a frequency of greater than 1 percent, and may be greater than 10 percent or 20 percent of a selected population. Polymorphic sites may be at known positions within a nucleic acid sequence or may be determined to exist. Polymorphisms may occur in both the coding regions and the noncoding regions (for example, promoters, introns or untranslated regions) of genes. Polymorphisms may occur at a single nucleotide site (SNP) or may involve an insertion or deletion as described herein. The SNPs are identified by their refSNP of dbSNP database of NCBI (http://www.ncbi.nlm.nih.gov/SNP).
As used herein “linkage disequilibrium” (LD) is the occurrence in a population of certain combinations of linked alleles in greater proportion than expected from the allele frequencies at the loci. Accordingly, if the genotype of a first locus is in LD with a second locus (or third locus etc.), the determination of the allele at only one locus would necessarily provide the identity of the allele at the other locus. Accordingly, two SNPs that have a high degree of LD may be equally useful in determining the identity of the allele of interest. Therefore, knowing the identity of the allele at one SNP is representative of the allele identity at another SNP in LD. Accordingly, the determination of the genotype of a single locus can provide the identity of the genotype of any locus in LD therewith and the higher the degree of linkage disequilibrium the more likely that two SNPs may be used interchangeably. When evaluating loci for LD those sites within a given population having a high degree of linkage disequilibrium (i.e. an absolute value for r2 is 0.5) are potentially useful in predicting the identity of an allele of interest (i.e. associated with the radio-sensitivity or the adverse effects). A high degree of linkage disequilibrium may be represented by an absolute value for r2>0.7 or by an absolute value for r>0.8. Additionally, a high degree of linkage disequilibrium may be represented by an absolute value for r2>0.85 or by an absolute value for r2>0.9 or by an absolute value for r2>0.95. In a preferred embodiment, a high degree of linkage disequilibrium is represented by an absolute value for r2 of 1. More particularly, when the absolute value for r2 is 1, two SNPs are interchangeable.
LD may be useful for genotype-phenotype association studies. For example, if a specific allele at one SNP site (e.g. “A”) is the cause of a specific clinical outcome (e.g. call this clinical outcome “B”) in a genetic association study then, by mathematical inference, any SNP (e.g. “C”) which is in significant LD with the first SNP, will show association with the clinical outcome. That is, if A is associated (˜) with B, i.e. A-B and C-A then it follows that C-B.
Tools are available for determining the SNP is linkage disequilibrium with another SNP. In particular, such data are available on the website www.broadinstitute.org/mpg/snap/.
Optionally, the linkage disequilibrium (e.g., an absolute value for r2) is calculated for a population selected from the group consisting of European super population, African super population and East Asian super population.
The SNPs having a linkage disequilibrium with an absolute value for r2 of at least 0.85 with rs3815496 can be selected in one of the following groups consisting of
The SNPs having a linkage disequilibrium with an absolute value for r2 of at least 0.85 with rs1131532 can be selected in one of the following groups consisting of
More particularly, item 1 relates to the European super population, item 2 to the African super population, item 3 to the East Asian super population and item 4 relates to the SNPs in common between European and African super populations.
In a preferred embodiment, the SNPs can be selected in the group consisting of rs9811673, rs9811639, rs3815496, rs3181143, rs3136606, rs3136607, rs3819773, rs1126161, rs63562861, rs1131542, rs11720451, rs9859809, rs6807069, rs6783667, rs9869255, rs9811650, rs9811646, rs62282578, rs1992868, rs1992869, rs6414542, rs1597086, rs9880799 and rs4894557. They have a linkage disequilibrium with an absolute value for r2 of at least 0.85 with both rs1131532 and rs3815496 and being in common between European and African super populations.
The methods, uses and kits according to the invention can be performed with one SNP as disclosed herein or with a combination thereof.
In a preferred embodiment, the subject is a human being. Optionally, the subject can belong to European super population, African super population or East Asian super population.
The method comprises determining the alleles of a SNP of the invention in a sample from the subject. It may comprise a previous step of providing a sample from the subject.
The term “sample”, as used herein, refers to a composition that is obtained or derived from a subject of interest that contains a cell or genetic materials suitable for genotyping a SNP. The source of the sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid. The sample may also be primary or cultured cells. Optionally, the sample can be obtained from a disease tissue/organ, especially from a cancer or tumor. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. In a preferred embodiment, the sample is saliva, urine, whole blood, plasma, or serum sample. The sample may be collected according to conventional techniques and used directly in the method of the invention or stored prior to its use. The sample may be treated prior to its use.
The allele of SNPs may be determined at the level of the DNA, or RNA by any method known by the skilled person.
These methods include, but are not limited to, direct sequencing-based methods, hybridization-based methods, primer extension-based methods, ligation-based methods, methods based on the conformation of a molecule containing the polymorphism, or invasive cleavage-based methods.
In a particular embodiment, the allele of SNPs is detected by direct sequencing using well-known techniques, preferably using next-generation sequencing technologies. The sequencing may be performed on the complete sequence of the TRAIL/TNFS10 gene, or more preferably on the genomic region containing rs3815496 and/or rs1131532.
In another embodiment, the allele of SNPs is detected by selective hybridization. Hybridization based genotyping methods include, but are not limited to, southern hybridization, genotyping on a microarray, in particular on a SNP microarray (amplification products are analysed by hybridization to target sequences immobilized on a solid support), methods using molecular beacons designed to only hybridize to a specific allele, and dynamic allele-specific hybridization (DASH) assay.
In another embodiment, the allele of SNPs is detected by selective amplification. Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), Touch-down PCR (TD-PCR), droplet-digital PCR (ddPCR), quantitative PCR (q-PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Preferred techniques use allele-specific PCR, tetra-primer ARMS-PCR (Shu et al., 2001, Nucleic Acids Res. 29 (17): e88).
The allele of SNPs may also be determined by enzyme based methods including, but not limited to, restriction fragment length polymorphism (RFLP) analysis, invader assay relaying on the activity of flap endonuclease that cleaves at specific nucleic acid structures, enzymatic digestion, TaqMan assay or 5′-nuclease probe assay relying on the 5′-nuclease activity of Taq DNA polymerase (e.g. implemented using droplet-digital PCR (ddPCR) or quantitative PCR (q-PCR)); oligonucleotide ligation assay, ligation rolling circle amplification or L-RCA (Xiaoquan et al., 2001, Nucleic Acids Res. 29 (22): e116), or primer extension based methods relying on the hybridization of a probe to the bases immediately adjacent to the specific polymorphic allele.
The allele of SNPs may also be determined using methods relying on the differences in conformation, weight, or size of molecules such as PCR-SSCP, MALDI-TOF mass spectrometry (Griffin and Smith, 2000, Trends in Biotechnology, 18: 77-84), denaturing-HPLC and temperature gradient gel electrophoresis (TGCE).
The various methods may be carried out in various reaction formats including homogeneous reactions and reactions on solid supports. Various detection methodologies may be employed in detecting SNPs including, but not limited to, radioactive detection, luminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, gel electrophoresis, and mass spectrometry.
Preferably, the allele of SNPs is determined using at least one nucleic acid probe hybridizing to a specific allele.
In a particular embodiment, the allele of the SNP rs1131532 is detected using a nucleic acid probe specifically hybridizing to the C allele and/or a nucleic acid probe specifically hybridizing to the T or G allele, at the polymorphic site rs1131532. Preferably, the allele at the polymorphic site rs1131532 is determined with a 5′-nuclease probe assay, using a nucleic acid probe specifically hybridizing to the C allele and/or a nucleic acid probe specifically hybridizing to the T or G allele, in particular using any probe described below as part of the kit of the invention.
In another particular embodiment, the allele of the SNP rs3815496 is detected using a nucleic acid probe specifically hybridizing to the A allele and/or a nucleic acid probe specifically hybridizing to the G allele, at the polymorphic site rs3815496. Preferably, the allele at the polymorphic site rs3815496 is determined with a 5′-nuclease probe assay, using a nucleic acid probe specifically hybridizing to the A allele and/or a nucleic acid probe specifically hybridizing to the G allele, in particular using any probe described below as part of the kit of the invention.
In another aspect, the present invention also relates to a kit comprising
(i) at least one nucleic acid probe capable of specifically binding or hydridizing to a SNP as disclosed herein, and/or
(ii) at least one set of primers suitable for use in amplification reaction, wherein the set of primers amplifies a SNP as disclosed herein,
and optionally, a leaflet providing guidelines to use such a kit.
The set of primers may allow amplification of the TRAIL/TNFSF10 gene in its entirety, or only a fragment of a SNP as disclosed herein.
In some embodiments, the nucleic acid probes may be attached to at least one reporter molecule generating a detectable signal. The detectable signal may be for example a fluorescent, luminescent or colored signal.
In preferred embodiments, the nucleic acid probes are attached to a fluorophore-quencher pair. These probes are particularly useful in 5′-nuclease probe assays. 5′-nuclease probes are single-stranded hybridization probes labeled with a donor-acceptor fluorophore pair that interact via FRET. The probe is designed to hybridize to its target DNA strand at the same time as the PCR primer. When Taq DNA polymerase extends the primer, it encounters the probe, and as a result of its 5′-nuclease activity, it cleaves the probe. Cleavage of the probe results in the separation of the donor fluorophore and quencher molecule, and leads to an increase in the intensity of the fluorescence signal.
A wide variety of fluorophore-quencher pairs are detailed in the literature (see .e.g. Marras et al. Methods Mol Biol. 2006; 335:3-16) and in supplier's catalogues.
Examples of fluorophores include, but are not limited to, fluorescein derivatives such as 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), tetrachlorofluorescein (TET) and hexachlorofluorescein (HEX), cyanine dyes such as Cyt, Cy3 and Cy5, Tetramethylrhodamine (TMR), Oregon Green dyes and Texas Red.
Examples of quenchers include, but are not limited to, DABCYL (4-(4-(dimethylamino)phenylazo) benzoic acid), QSY™ quenchers (Invitrogen, Carlsbad, Calif.), ECLIPSE™ quenchers (Epoch Biosciences, Bothell, Wash.) and BHQ™ (BHQ-1, 2, and 3 (Biosearch Technologies, Inc. Novato Calif.)) and DDQ-1 or -2.
In particular embodiments, the fluorophore-quencher pair is selected from HEX-BHQ1 and 6-FAM-BHQ1. Preferably, distinct fluorophore-quencher pairs are attached to each nucleic acid probe type, i.e. probes specific to a first allele and probes specific to the other allele for a specific SNP as disclosed herein, in order to distinguish their signals.
In particular, the nucleic acid probe specific to a first allele of a SNP as disclosed herein may comprise hexachlorofluorescein attached to its 5′ end and BHQ1 quencher attached to its 3′ end. The nucleic acid probe specific to the other allele of the SNP may comprise 6-carboxyfluorescein attached to its 5′ end and BHQ1 quencher attached to its 3′ end.
The kit may also comprise at least one molecular beacon or a microarray that can be used to detect a SNP as disclosed herein.
The kit may further comprise one or several reagents for detecting the hybridization of said at least one nucleic acid probe and/or for amplifying and/or detecting said genetic alteration or polymorphism site. In a particular embodiment, the kit further comprise reagents needed to perform droplet digital PCR.
The present invention also relates to the use of this kit for assessing the risk of developing adverse effects after radiotherapy in a subject, said adverse effects being acute and/or subacute dermatitis, and/or lymphocyte apoptosis, or for determining the radiation dose suitable for a subject, according to the method of the invention.
All embodiments disclosed above for the method of the invention are also contemplated in this aspect.
The present invention also takes advantages of the TRAIL modulation.
Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting.
The sensitivity of subpopulations of human T-lymphocytes to ionizing radiation-induced apoptosis was quantified eighteen hours after irradiation (0-2 Gy) of PBMC samples of healthy blood donors using the previously defined radio sensitivity assay based on immunophenotyping and AnnexinV-labeling. Whereas the CD62L-positive T lymphocyte subpopulations did not undergo apoptosis (
Only 1 out of 45 studied samples showed discordant T4EM lymphocyte radio sensitivity and TRAIL/TNFSF10 expression level.
To study the effects of irradiation on TRAIL- and TRAIL-receptors mRNA levels in T4EM lymphocytes, the inventors performed multiplexed determination of their mRNA levels on independent triplicate sorts of T4EM lymphocytes from “sensitive” and “resistant” samples at 2, 4, 6 and 24 hours after 2 Gy irradiation. Expression of the decoy receptor TNFRSF10c/DcR1 was below detection limit in T4EM lymphocytes (data not shown). No significant effect of irradiation on TRAIL/TNFSF10, on decoy receptor TNFRSF10d/DcR2 (not shown), and on pro-apoptotic receptor TNFRSF10a/DR4 mRNA levels was found in “sensitive” or “resistant” samples (
TRAIL can be soluble (sTRAIL) or membrane-bound (mTRAIL)(Schneider et al., 1998, J. Exp. Med. 187:1205-1213; Wajant et al., 2001, Oncogene. 20:4101-4106). Under the experimental conditions used for radiation-induced apoptosis of T4EM lymphocytes, the levels of sTRAIL in media were below sandwich ELISA detection level (<20 pg/ml; data not shown). The inventors thus focused their study on mTRAIL. Using a monoclonal antibody panel comprising CD253, they measured the level of cell-surface mTRAIL expression by flow cytometry. A four-fold higher expression of mTRAIL was found on T4EM lymphocytes of “sensitive” compared to “resistant” samples (
A ten-fold increase in cell concentration did not increase apoptosis-induction, irrespective of sample sensitivity-classification. On a set of 9 “sensitive” samples, a ten-fold higher cell concentration even resulted in 1.5-fold lower apoptosis-induction in T4EM lymphocytes (
Shedding of mTRAIL depends on metalloprotease activity. The inventors thus studied the effect of 1,10-phenanthroline, a broad-range metalloprotease-inhibitor (Yan et al., 2011, CORD Conference Proceedings. 108-111). Two hours after addition of 1,10-phenanthroline, an increase of mTRAIL expression was observed in T4EM lymphocytes in “resistant” and “sensitive” samples (
The increased mTRAIL expression, that was associated with an increased expression of proapototic TRAIL-receptor DR5 (data not shown), was associated with a rapid induction of apoptosis (
To investigate any genetic association between the TRAIL/TNFSF10 locus and radio sensitivity of T4EM lymphocytes, selected regions in the coding- and flanking regions of the TRAIL/TNFSF10 gene that contain SNPs that might be associated to- or act as eQTLs, were genotyped. A 4.2 kb region encompassing the TRAIL/TNFSF10 gene from 373 individuals previously characterized for the T4EM lymphocytes radio sensitivity and subjected to heritability and segregation analysis (Schmitz et al., 2007, Int J Radiat Oncol Biol Phys. 68:1169-1177) was sequenced. Table 1 summarizes results for the 36 polymorphic markers identified, with their frequencies and positions in the studied population. Single marker association analysis between the 15 identified frequent variants (MAF>5%) and T4EM lymphocytes radio sensitivity by Family-Based Association Testing (FBAT) showed a significant association for 3 SNPs; rs3815496 (p=0.03; intron 3, MAF=0.28), rs1131532 (p=0.04; exon 5, MAF=0.28), and rs1131535 (p=0.05; 3′ UTR exon 5, MAF=0.38). SNPs rs1131532 and rs3815496 were in strong linkage disequilibrium, and in partial linkage with rs1131535 (r2 of less than 0.5, specifically 0.49 and 0.47, respectively). Several other SNPs in the sequenced region were found to be marginally but significantly associated (Table 2). Multi-Marker association testing between the 15 most frequent SNPs of the TNFSF10/TRAIL gene and T4EM lymphocytes radio sensitivity was shown significant by the Family based MM association test (FBAT-MM) (p=0.02) and by Fisher product (p=0.04), demonstrating a genetic contribution to the T4EM lymphocytes radio sensitivity in or close to the TRAIL/TNFSF10 gene. The analysis of a selection of 126 unrelated individuals, using T4EM lymphocytes radio sensitivity median values by genotype at rs3815496 identified A as the risk allele for high levels of radiation-induced apoptosis in T4EM lymphocytes (
Association between the identified TRAIL/TNFSF10 SNPs and CD8+ lymphocyte apoptosis as well as with clinical radio sensitivity endpoints was studied in a set of 113 genotyped breast cancer patients, included in the Co-Ho-RT study (Azria et al. 2010, The Lancet Oncology, 11 (3), 258-265). Blood samples from these patients are characterized for radiation-induced CD8+ T-lymphocyte apoptosis, according to Ozsahin et al., 2005 (Clin Cancer Res. 11:7426-7433). Genetic association study indicated a trend towards association of the three SNPs, rs3815496 (MAF=0.28, p=0.06), rs1131532 (MAF=0.28, p=0.06), and rs1131535 (MAF=0.44, p=0.09), with radiation-induced CD8+ T-lymphocyte apoptosis. Finally, acute and subacute dermatitis (10 cases), acute erythema (44 cases), acute hyperpigmentation (7 cases), late fibrosis (28 cases), and late telangiectasia (3 cases) were evaluated for association with these three TRAIL/TNFSF10 SNPs. None of these three SNPs was associated with late fibrosis, acute erythema, or acute hyperpigmentation (Table 3). An association was shown with late telangiectasia (Table 3) but, due to insufficient number of cases this result will not be considered. Interestingly, for acute and subacute dermatitis, a strong association was identified with rs3815496 (p=0.047, effect allele=A, OR: 3.88, 95% c.i: 0.87-17.22), and with rs1131532 (p=0.045, effect allele=C, OR 3.90, 95% c.i: 0.88-17.30), but not with rs1131535.
Advances in radiotherapy treatment suffer from the lack of validated tests and genetic markers that can be used to predict individual radio sensitivity. Human T4EM lymphocyte radiation induced apoptosis indicates that expression of TRAIL/TNFSF10 in resting T4EM lymphocytes identifies individuals with a high apoptotic response to ionizing radiation of these lymphocytes in vitro. This T4EM lymphocytes sensitivity can be modulated by anti-TRAIL/CD253 as well as by exogenous sTRAIL, and T4EM lymphocytes apoptosis involving TRAIL signaling could be induced in vitro by inhibition of matrix-metalloproteases. Finally, the inventors identified the association between three SNPs within the TRAIL/TNFSF10-gene and T4EM lymphocytes radio sensitivity, a quantitative trait that had been previously shown to display significant heritability and the segregation of which showed compatible with a Mendelian mode of transmission. A role of TRAIL in the clinical response to radiation and in its physiopathology is suggested by the association between the alleles related to higher apoptosis for two of these three TRAIL/TNFSF10 SNPs and acute and subacute dermatitis, a subset of radiotherapy induced skin reactions (RISR) in a cohort of breast cancer patients. Amongst the identified SNPs, rs1131532 was previously described to be associated with the concentration of the systemic inflammatory biomarker P-selectin (Schnabel et al. 2009, Circulation: Cardiovascular Genetics, 2 (3), 229-237). The inventors did not identify association of TRAIL/TNFSF10 SNPs with acute erythema, or with acute hyper-pigmentation.
The inventors did not detect association between the listed TRAIL/TNFSF10 SNPs and late fibrosis, although a trend exists between the TRAIL/TNFSF10 SNPs and the CD8+ T-lymphocyte apoptosis phenotype according to Ozsahin. Late fibrosis is a severe complication of radiotherapy that has been previously related to lymphocyte resistance to apoptosis. Association between low apoptosis of CD8 lymphocytes and late fibrosis was first evidenced by Ozsahin et al. (Ozsahin et al., 2005, Clin Cancer Res. 11:7426-7433) and replicated in several independent studies. The radio sensitivity phenotyping strategy employed in the present study is markedly different in that it relies on L-Selectin/CD62L-negative CD4 positive lymphocytes only, specifically excluding apoptosis-resistant L-Selectin-positive lymphocytes. It may be relevant to study inter-individual differences in L-selectin shedding/down regulation following radiation to further investigate the association between lymphocyte apoptosis, radio sensitivity, and late toxicity. Altogether the present study highlights a function of TRAIL in lymphocyte radiation induced apoptosis associated with TRAIL/TNFSF10 SNPs. International consortium research that will provide the power of GWAS will enable to appreciate the relative contribution of the identified TRAIL/TNFSF10 SNPs to radiotherapy toxicity (Kerns et al., 2013, Radiother Oncol. 107:372-376).
All samples were prepared from venous blood drawn from healthy normal volunteers between 9:00 am and 11:00 am. The inclusion of healthy volunteers was preceded by a medical interview and informed written consent was obtained from all donors, in accordance with local ethics guidelines at Fondation Jean Dausset-CEPH or Etablissement Francais du Sang, Hopital Saint Louis, Paris.
Peripheral blood mononuclear cells (PBMC) were prepared from fresh (within 6 h, average 180 minutes) anticoagulated blood samples, by standardized procedures. Briefly, 30 ml of two-fold diluted blood were underlayered with 10 ml of Histopaque®-1077 (Sigma-Aldrich), and centrifuged at 2,000 g for 20 minutes. The collected interlayer was washed twice with 40 ml of RPM11640/15% Fetal Calf Serum (FCS), and used as further described or collected in FCS/10% DMSO for cryopreservation. Fractions of 1 ml each, corresponding to 4 ml of blood, were immediately frozen, using controlled temperature decrease, and stored in liquid nitrogen. Thawing of cells was performed by rapid transfer of cryotubes to 56° C. waterbath (45 sec), addition of 1 ml of FCS and transfer of contents to 12 ml of RPMI/15% FCS. Thawed cells were washed twice, to be finally resuspended in the appropriate volume of culture medium (RPM11640/15% FCS).
DNA extractions were performed from PBMC by classical phenol/chlorophorm procedures. Yield and purity were assessed spectrophotometrically at 260 and 280 nm (Thermo Scientific NanoDrop™ 1000). Familial relations were recorded from oral interview and HLA-A sequencing was used to exclude samples presenting incompatibilities with announced familial interrelations from family based association analysis.
All fluorochrome-coupled monoclonal antibodies were used at saturating conditions, as determined by titrations. Anti-human CD antibodies came from Becton Dickinson ImmunoCytometry Systems (CD62L-PE, CD62L-PECy5.1, CD14-PECy5.1, CD-235a-PECy5.1, CD27-PECy5.1, CD4-PECy7, CD19-PECy7, CD45RA-APC, CD8-APCH7), from Beckman Coulter (CD3-PETxR) from Biolegend (CD253/TRAIL-PE, CD62L- BV421, CD3-BV605), or from R&D Systems (CD262/DR5-APC). Recombinant human soluble TRAIL (rh-sTRAIL) and recombinant human soluble DR5 (rh-s-TRAIL was from R&D Systems, AnnexinV-FITC from Becton Dickinson, and 1,10-phenanthroline from Sigma-Aldrich.
Cell suspensions at approximately 5×105 cells/ml (or at cell densities specified in the results section) in RPM11640/15% FCS, were irradiated at a dose rate of approximately 0.5 Gy/min with a 137Cs-source (IBL637; Cis-Bio international/Scherring, Saclay, France), where incubated in the presence of indicated concentrations of recombinant human soluble TRAIL (rh-sTRAlL), recombinant human soluble DR5 (rh-sDR5), anti-TRAIL cytotoxicity-blocking antibody (CD253; clone RIK-2), or of 1,10-phenanthroline (stock solution of 200 mM in DMSO). All experiments described were performed with the same batch of heat inactivated FCS, previously tested for compatibility with our experimental procedures. Incubations of 18 hours or less as specified in results section were at 37° C. and 5% CO2.
Treated cells were collected and washed with AnnexinV buffer (140mM NaCl, 5mM CaCl2, 10 mM HEPES; ph7.4), and stained with 2.5 μl AnnexinV-FITC in combination with fluorochrome-conjugated CD markers in 100 ul for 30 min. For cell sorting for gene expression analysis, Annexin V positive cells were excluded, and CD3 labeling was omitted to avoid TCR-mediated activation signaling. Furthermore, CD14 and CD235a were included to exclude monocytes and reticulocytes susceptible to contaminate the sort gates. CD27 was included for sorts of cryopreserved samples to avoid contamination of sort gates by central memory lymphocytes that transiently lost CD62L through sample manipulation procedures. For CD253 (mTRAIL) quantification, a compact panel designed to limit the need for compensation including AnnexinV, CD253, CD3, CD4, and CD62L was used.
Cells were washed in AnnexinV-buffer and just prior flow cytometry, 50 μL of AnnexinV-buffer containing 0.2 μg/mL Hoechst 33258 (Molecular Probes, Eugene, Oreg.), was added for dead cell exclusion. Samples were analyzed using a three-laser CyAn LX system, (DakoCytomation, Fort Collins, Colo.) or a 5-laser SORP LSRII (Becton Dickinson). Cell sorting was performed using a custom-built five-laser InFlux system (Becton Dickinson). All filters were from Chroma Technologies Inc., Brattleboro, Vt. All fluorescence signals were log-amplified, and stored in listmode. CompBeads (Becton Dickinson) were used to assist in determination of spill-over in every individual experiment (CD3-FITC was used instead of AnnexinV-FITC) and Fluorescence Minus One (FMO) controls were used where needed, in particular for mTRAIL quantifications (SortWare; Becton Dickinson or Flowjo version 9.6.4; Treestar, Ashland, Oreg.). All sorts were performed in single droplet count mode, with an extended coincidence mask of one-and-a-half droplet. Doublet exclusion was performed by gating on the triggering parameter (FSC) versus time of flight.
Data was analyzed using Flowio. Listmode file analysis proceeded through the exclusion of non-lymphocyte events, predominantly monocytes, by using a large scattergate, shaped to include apoptotic lymphocytes (lower FSC, higher SSC). Subsequently, dead cells were excluded on the basis of HO33258 fluorescence. T Lymphocytes were identified on the basis of positive PETxR (CD3) fluorescence. A PECy7 (CD4) versus APCH7 (CD8) histogram allowed identification of CD4-positive T lymphocytes (T4) and CD8-positive T lymphocytes (T8). An APC (CD45RA) versus PE (CD62L) histogram discriminated between naive (CD62L+CD45RA+), central memory (CM; CD62L+CD45RA−), effector memory (EM; CD62L-CD45RA-), and terminal effector (TE; CD62L-CD45RA+) T4 and T8. The proportion of apoptotic cells was determined by application of an identical gate on a bivariate plot of FSC versus FITC fluorescence (AnnexinV) to all of the identified subpopulations. Dose-effect curves were generated using at least four doses, and used for the quantitative evaluation of 1,10-phenanthroline sensitivity and radiation sensitivity, as previously described (Schmitz et al., 2003, Int J Radiat Oncol Biol Phys. 57:769-778).
Cells of interest, recovered by centrifugation directly after sort, were lysed by RLT buffer (Qiagen) and cryopreserved until RNA extraction. Total RNA was extracted from a minimum of 300,000 cells using the RNeasy Plus Micro Kit (Qiagen). RNA samples were re-suspended in RNase-free water and stored at −80° C. RNA yield and purity were assessed spectrophotometrically at 260 and 280 nm (Thermo Scientific NanoDrop™ 1000). RNA integrity was evaluated using a Bioanalyzer 2100 (Agilent). Microarray experiments and part of data analysis were performed by PartnerChip (S. B., Evry, France) following the Affymetrix-recommended procedures. Target was prepared from 4 sensitive and 4 resistant individuals. Targets were pooled after individual quality control in equal proportions and hybridized on a HU133 2.0 plus array (54645 probe sets) according to the Affymetrix Two-Cycle technical protocol. Fluorescent images were detected in a GeneChip Scanner 3000 (Affymetrix). Expression data and raw expression data (CEL files) were generated using GCOS software (Affymetrix). Quality control was assessed based on 3′/5′ ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin control probe sets.
Triplicate sorts for qPCR gene expression studies were performed by direct deposition of cells in MicrAmp optical tubes (Applied Biosystems), containing 15 μl of one step RT-qPCR mix. RT-qPCR pre-amplification mix contained Platinum Taq polymerase and SuperScript III reverse transcriptase (Invitrogen), a mixture of Taqman primer-probes, specific for the transcripts of interest (Table 4; Applied Biosystems) at 0.2× concentration, CellsDirect One-Shot qRT-PCR buffer (Invitrogen), and Superaseln RNase inhibitor. Immediately following cell sorting, samples were thoroughly mixed, and reverse transcribed (55° C. for 10 min, 50° C. for 50 min), and pre-amplified (14 cycles of PCR at 95° C. for 15 sec and 62° C. for 45 sec). After pre-amplification, samples were diluted ten-fold, for gene-specific quantitative PCR (35 cycles of 95° C./15 sec, 62° C./45 sec) on a HT7900 (Applied Biosystems). All primers were standard Taqman assays (Applied Biosystems), and reaction conditions were according to manufacturers indications. Results were expressed as the difference between the Ct of the internally amplified reference gene (VIC-labeled RPLPO, ACTB, or GAPDH), and the FAM-labeled test gene, averaged over triplicate sorts, and termed dCT. Assay performance control on cryopreserved lymphocytes included mean dCt on different cryopreserved vials from same blood sample, mean dCt from independent blood samples from same individual. Samples from reference individuals were included in each experiment. Radiation induction of expression is expressed as the difference between dCT at dose 2.0 Gy and dCt at dose 0 Gy, for each timepoint after irradiation, and was termed ddCT.
Primers were established in order to amplify by PCR the exon-containing DNA fragments and the promoters. The PCRs were performed in a 15 μL reaction mixture containing 25 ng of DNA (lists of primers Tables 5 and 6). Sequencing reactions were performed according to the dye terminator method using an ABI PRISM® 3730xl DNA Analyzer (Applied Biosystems, Foster City, Calif., USA). Alignment of experimental results, SNP discovery and genotyping were performed with the software Genalys. The genomic sequence used for the alignment is TRAIL/TNFSF10 (NC_000003.11). The genotypes for rs3136597 and rs2241063 were obtained by Taqman technology, assays C_27464917_20 and C_3260973_20 respectively (Applied Biosystems).
The FBAT method and software (Laird et al., 2000, Genet Epidemiol. 19 Suppl 1:S36-42; Rabinowitz and Laird, 2000, Hum Hered. 50:211-223) was used to perform a single marker analysis between the frequent SNPs and the quantitative trait while taking into account the family structure (command fbat-o). Two methods have been used to obtain a p-value for the global association of TNFSF10 with the trait : a) The Fisher product (Fisher, 1925, Statistical Methods for Research Workers.) of the p-values obtained from the single point association analysis was taken, and its significance was assessed using 10,000 permutations, b) The FBAT Multi-Marker statistic (Rakovski et al., 2007, Genet Epidemiol. 31:9-17; Chen et al., 2013, Genet Epidemiol. 37:196-204) was computed, and its significance was assessed using 10,000 permutations.
Acute and subacute dermatitis, acute erythema, acute hyperpigmentation and late fibrosis were assessed prospectively using the CTCAE v3 grading system in the CO-HO-RT trial (Azria et al. 2010, The Lancet Oncology, 11 (3), 258-265). Toxicity was treated as a binary outcome and was determined by taking the highest CTCAE grade occurrence during radiotherapy to 6 weeks after radiotherapy for acute toxicities and from 3 months after radiotherapy to the end of follow-up (minimum 2 years, maximum 8 years) for late toxicities. For each toxicity endpoint, patients were considered cases if they had CTCAE grade ≥2 and controls if they had CTCAE grade 1. The percentage of CD8+ lymphocytes undergoing radiation-induced apoptosis was treated as a continuous outcome.
Genomic DNA was isolated from blood, genotyped using a commercial genome-wide SNP array (Affymetrix SNP6.0, Affymetrix, San Diego, Calif.)]. Because rs1131532, and rs3815496 were not directly genotyped on the array, the surrounding region of chromosome 3 (position 100,000,001 to 200,000,000, hg build 19) was imputed to the 1000 Genomes reference data using IMPUTE2 software [PMID: 19543373]. Following imputation, the SNPs of interest were confirmed to be in Hardy-Weinberg equilibrium (p-value>10−6) and showed good imputation quality (info score>0.3). SNPTEST software [PMID: 17572673] was used to analyze SNP-phenotype association with the frequentist test and expected method, which uses expected genotype counts (i.e. genotype dosages) obtained from imputation. An additive genetic inheritance model was assumed in all analyses.
Statistical analyses and data presentation were performed with Stata (Stata Corp., College Station, Tex.). In box plot representations of results, boxes extend from 25th to 75th percentiles, with whiskers indicating upper- and lower adjacent values (1.5 times interquartile range). Each box represents at least three independent experiments.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15306324.3 | Aug 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/070153 | 8/26/2016 | WO | 00 |
