Method for molecular diagnosis of rare molecular subtypes of gastrointestinal stromal tumors (GIST)

Abstract

The present invention relates to a method for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, comprising the steps of: providing at least one isolated tissue sample comprising at least one nucleic acid and the subsequent extraction of the at least one nucleic acid. The method according to the present invention further comprises a step of quantifying the at least one previously isolated nucleic acid. Furthermore, the method according to the present invention comprises preparing at least one amplicon library of said previously isolated nucleic acid, which comprises sequences obtained from the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes. This step is followed by the step of purifying the at least one amplicon library and the step of simultaneously sequencing the amplicons themselves. The present invention also relates to a multi-gene panel for ex vivo molecular diagnosis of gastrointestinal stromal tumors and a kit comprising such a panel.

Description

[0001] “METHOD FOR MOLECULAR DIAGNOSIS OF RARE MOLECULAR SUBTYPES OF GASTROINTESTINAL STROMAL TUMORS (GIST)”

[0002] TECHNICAL FIELD

[0003] The present invention is in the technical field of gene detection and relates specifically to a method for detecting gastrointestinal stromal tumors, based on the NGS method.

[0004] Specifically, the present invention concerns a method for ex vivo molecular diagnosis of very rare molecular subtypes of gastrointestinal stromal tumors (GISTs).

[0005] The present invention also concerns a multi-gene panel usable in NGS methods, for ex vivo molecular diagnosis of very rare molecular subtypes of gastrointestinal stromal tumors (GISTs). The present invention further concerns a kit for ex vivo molecular diagnosis of rare gastrointestinal stromal tumors of gastrointestinal stromal tumors (GISTs).

[0006] STATE OF THE ART

[0007] Gastrointestinal stromal tumors, referred to by the acronym GIST (Gastro Intestinal Stromal Tumors), are rare neoplasms that can develop throughout the gastrointestinal tract, from the esophagus to the rectum.

[0008] In most cases, about 90%, these neoplasms are determined by mutations in the sequence of specific genes. Specifically, these mutations are involved in the activation of genes KIT (protooncogene receptor tyrosine kinase), PDGFR-a (Plateled Derived Growth Factor Receptor A), NRAS (NRAS proto-oncogene, GTPase), KRAS (KRAS proto-oncogene, GTPase), BRAF (B- Raf proto-oncogene, serine / threoninekinase).

[0009] Mutations involving these genes are known and easily identified by diagnostic techniques already known in the state of the art.

[0010] However, in the remaining 10% of cases, the KIT and PDGFR-a genes have no mutation in their gene sequence. In these cases, they are referred to as WT GIST diseases, or Wild Type Gastro Intestinal Stromal Tumors.

[0011] WT GISTs are a family of diseases each of which may have different alterations at the level of the SDHA (succinate dehydrogenase complex flavoprotein subunit A), SDHB (succinate dehydrogenase complex flavoprotein subunit B), SDHC (succinate dehydrogenase complex flavoprotein subunit C), SDHD (succinate dehydrogenase complex flavoprotein subunit D), NF1 (neurofibromin 1) and FGFR (fibroblast growth factor receptor) genes.

[0012] In recent years, the widespread use of gene sequencing techniques based on NGS (Next Generation Sequencing) technology as a diagnostic method in oncology field has enabled the diagnostic procedure for this type of cancer to be improved.

[0013] Actually, identification of the specific gene mutations responsible for oncological diseases, as in the case of WT GIST, is crucial for a correct and timely diagnosis and subsequent choice of the most appropriate medical therapy.

[0014] However, the mutations involved in the onset of WT GIST diseases are very difficult to detect even with the latest NGS sequencing techniques.

[0015] For example, molecular genetic studies on the SDHA gene are difficult to implement because this gene has numerous highly homologous pseudogenes.

[0016] The presence of numerous highly homologous pseudogenes complicates the step of amplifying the target gene sequence by polymerase chain reaction, as said reaction simultaneously amplifies not only the target gene but also a pseudogene having a nucleotide sequence very similar to the target one.

[0017] Unwanted amplification of pseudogenes generates a large amount of data (background noise), which complicates the interpretation of the data obtained and makes the diagnosis inaccurate.

[0018] Molecular genetic studies on the NF1 gene are also difficult to implement because this gene has a large number of exons (59 exons) and its nucleotide sequence is subject to intragenic deletion or rearrangement events.

[0019] It is known in the relevant field a genomic test dedicated to GISTs without KIT and PDGFR-a mutations, in which RAS and BRAF genes but also numerous other genes that are not relevant in diagnosing WT GIST oncological diseases are taken into account.

[0020] The large number of genes investigated greatly reduces the ability of this type of genomic test to correctly resolve the mutational status of WT GIST cases, that is, to determine which mutations led to the onset of the disease.

[0021] To date, in order to obtain an accurate diagnosis of WT GIST for KIT, PDGFR-a, KRAS and BRAF genes, that is, in the case where such genes do not have gene mutations, it is necessary to perform the study of numerous individual genes by targeted sequencing.

[0022] The sequencing methods, for targeted study, of WT GIST cases for KIT / PDGFRA / KRAS / BRAF currently have numerous drawbacks.

[0023] The main drawback of sequencing methods currently commercially available or in the research field lies in the fact that they require very long lead times because they involve the preparation and execution of multiple tests, each investigating a single alteration and being performed at different times.

[0024] Another drawback of currently available sequencing methods lies in the fact that they allow only one sample to be analyzed at a time.

[0025] An additional drawback of the currently available sequencing methods lies in the fact that the accuracy of the data obtained with respect to the SDHA and NF1 genes is rather low, since they are affected, for example, by the unwanted sequencing of pseudogenes.

[0026] Another drawback of currently available sequencing methods lies in the fact that setting up and running multiple tests results in high implementation costs.

[0027] Another drawback of currently available sequencing methods lies in the fact that setting up and running multiple tests results in higher reagent consumption.

[0028] An additional drawback of currently available sequencing methods lies in the fact that they require significant input of skilled labor.

[0029] OBJECTS OF THE PRESENT INVENTION

[0030] Object of the present invention is to provide a method for ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes, from isolated biological samples.

[0031] A further object of the present invention is to provide a method for ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes, that allows simultaneously characterizing all possible potentially identifiable alterations in WT GISTs.

[0032] Still object of the present invention is to provide a method for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes, that exhibits a high degree of accuracy, even in the case where the target gene has numerous pseudogenes having a nucleotide sequence very similar to the target gene.

[0033] A further object of the present invention is to provide a method for ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes, that allows simultaneously analyzing multiple samples.

[0034] Still object of the present invention is to provide a method for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes, that exhibits low implementation costs.

[0035] In addition, object of the present invention is to provide a method for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes, that allows to obtain results in a short time.

[0036] Furthermore, object of the present invention is to provide a method for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes, that require the use of a limited amount of skilled labor.

[0037] Further object of the present invention is to provide a multi-gene panel for ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes, that is, in the case where these genes do not have mutations. Still object of the present invention is to provide a kit for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes.

[0038] DESCRIPTION

[0039] The aforementioned objects, as well as the other objects, are achieved by the subject matter of the present invention, that is, a method for ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly for molecular diagnosis of gastrointestinal stromal tumors not caused by mutations in KIT, PDGFRA, KRAS, and BRAF genes. The method object of the present invention comprises the steps of: a) Providing at least one isolated sample comprising at least one nucleic acid; b) Extracting said nucleic acid; c) Quantifying said previously isolated nucleic acid; d) Preparing at least one amplicon library of said previously isolated nucleic acid, said at least one amplicon library comprising sequences obtained from the FGFR1, NF1, SDHA, SDHB, SDHC andSDHD genes; e) Purifying said at least one amplicon library; f) Parallel sequencing said amplicons.

[0040] Advantageously, the method according to the present invention allows the ex vivo diagnosis of gastrointestinal stromal tumors, particularly the diagnosis of those GIST tumors not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes.

[0041] Advantageously, the method according to the present invention allows the diagnosis of those GIST tumors caused, in particular, by rare gene mutations in FGFR1, NF1, SDHA, SDHB, SDHC or SDHD genes.

[0042] The complexity of these genes makes the detection of rare mutations occurring in their nucleotide sequence very complicated.

[0043] To ensure a diagnosis with an acceptable degree of accuracy, traditional diagnostic methods must be applied to FGFR1, NF1, SDHA, SDHB, SDHC or SDHD genes taken individually.

[0044] Advantageously, the method according to the present invention allows the simultaneously detection of all the possible gene alterations associated with GIST tumors not caused by mutations in KIT / PDGFRA / KRAS / BRAF genes.

[0045] In addition to allowing the simultaneous detection of multiple gene mutations, the method object of the present invention advantageously exhibits a high degree of accuracy, especially in the case where the target gene has numerous pseudogenes having a nucleotide sequence very similar to the target gene.

[0046] Advantageously, the method according to the present invention allows multiple samples to be analyzed simultaneously.

[0047] Furthermore, simultaneous detection of rare mutations in multiple samples allows costs to be reduced and a limited number of skilled labor to be employed.

[0048] Step a) of the present diagnostic method involves providing at least one isolated sample comprising a nucleic acid.

[0049] In embodiments, the isolated sample is an isolated tissue sample.

[0050] For example, the tissue can be selected from tissue fixed in formalin and embedded in paraffin (FFPE) or fresh / frozen tissue.

[0051] In embodiments, said isolated tissue sample is a tissue fixed in formalin and embedded in paraffin (“FFPE”, or “Formalin-fixed, paraffin-embedded”). Isolated tissue samples fixed in formalin and embedded in paraffin can be obtained by fixation protocols well known in the field of anatomic pathology.

[0052] In an embodiment of said method, said at least one sample is a tissue section. In embodiments, a plurality of samples is provided, for example, a plurality of isolated tissue samples fixed in formalin and embedded in paraffin, for example, 2 or 3 tissue samples.

[0053] In embodiments, the isolated sample comprising at least one nucleic acid is a tissue section fixed in formalin and embedded in paraffin, preferably having a thickness ranging from 5 pm to 10 pm, preferably 10pm.

[0054] In particular, samples can advantageously be obtained from isolated tissues in which the absence of mutations in KIT, PDGFRA, KRAS, and BRAF genes has already been determined. In more detail, said isolated tissue samples contain at least one nucleic acid, specifically DNA. More preferably, said nucleic acid is double-stranded DNA (dsDNA).

[0055] Step b) of the present diagnostic method involves extracting said at least one nucleic acid from said sample.

[0056] Said extracting step is performed by extraction protocols already known, per se, in the art.

[0057] For example, nucleic acid extraction can be carried out by manual extraction kits using columns of different matrices and / or extraction protocols that take advantage of automated extractors. Preferably, any extracting method per se already known can be applied for nucleic acid extraction in said step b) of the present diagnostic method.

[0058] Preferably, said extracting step b) involves DNA extraction.

[0059] Extracting step b) is followed by step c) of quantifying said at least one nucleic acid isolated in step b), which is performed by techniques known, per se, to the skilled in the art.

[0060] Preferably, said quantification techniques comprise, for example, methods such as Real Time PCR.

[0061] Next, said isolated and quantified nucleic acid, in this case DNA, is used, according to step d) of the method according to the present invention, for the preparation of at least one amplicon library.

[0062] An amplicon library consists of specific portions of the genome amplified by PCR and conjugated with adapters, that is, specific sequences located at the ends of the amplified fragment.

[0063] According to the present invention, the library of amplicons used in the method of the invention comprises sequences obtained from the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes. Preferably, the library(s) of amplicons is constituted by the sequences obtained from the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

[0064] In other words, in embodiments, the library(s) of amplicons does not comprise sequences obtained from genes other than FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

[0065] In embodiments, the library comprises or, alternatively, is constituted by, the amplicons set forth in Table 1 below.

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] The obtained amplicons will then be sequenced, preferably by NGS (Next Generation Sequencing) sequencing.

[0083] The step of preparing said amplicon libraries is an important step for parallel NGS sequencing.

[0084] Generally, amplicon-based libraries for NGS are made by polymerase chain reaction (PCR); in particular, such technology allows the amplification of a very large number of target genomic regions that will constitute the library itself. The amplification protocol used is as follows:

[0085] 1. 99°C for 2'

[0086] 2. 99°C for l5"

[0087] 3. 60°C for 4 minutes

[0088] Steps 2-3 are repeated 19-21 times (preferably 20 times).

[0089] In the case of the present invention, said amplicon libraries can be made, for example, by means of the Ion AmpliSeq Library Kit Plus kit (Thermo Fisher Scientific) equipped with two different primer pools for the PCR reaction.

[0090] Preferably, said step of preparing said libraries involves several sub-steps.

[0091] In embodiments, the first step involves generating a mixture comprising an amount of nucleic acid, preferably DNA, preferably between 10 ng and 50 ng, more preferably ~40 ng.

[0092] Said master mixture further comprises nuclease-free water.

[0093] In embodiments, in a next step, said solution is divided into two wells of a PCR reaction plate. A 5 ul amount of primers related to the regions denoted by the number " 1" in the “Primer Pool” column (Table 1) is then added to one of said wells.

[0094] A 5 ul amount of primers related to the regions denoted by the number "2" in the “Primer Pool” column (Table 1) column is added to the second of said wells. This subdivision allows the possibility of forming "primer dimers" (i.e., products of nonspecific alignment events and primers during the PCR reaction) to be minimized. Specifically, said first and said second primers consist of a short DNA sequence configured to promote the initiation of the polymerization reaction for the amplification of the target DNA sequences to be sequenced.

[0095] In the present case, the preparation of at least one amplicon library is carried out by using primers adapted to produce amplicons referring to the genomic sequences of FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

[0096] Primers adapted to produce amplicons from the genomic sequences of the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes can be obtained by techniques known, per se, in the art.

[0097] In embodiments, the next step of said library preparation step involves amplifying target DNA regions.

[0098] In embodiments, target DNA regions are obtained from the sequence of genes involved in the onset of GIST tumors, particularly from genes involved in the onset of WT GIST tumors.

[0099] Specifically, in embodiments, said target regions are obtained from the sequences of the following genes associated with rare molecular subtypes of GIST tumors, that is, from the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

[0100] The diagnostic method that is object of the present invention involves the preparation of a library comprising all the sequences of said genes.

[0101] The amplification reaction of target regions by PCR involves the addition of the polymerase enzyme to said reaction solutions.

[0102] Next, the steps of denaturing, pairing and extending are performed in succession. Preferably, denaturation is performed at a temperature of 99°C for 15 seconds, so as to promote the opening of the DNA double helix and activate the polymerase enzyme. The pairing step, during which the primers bind to the complementary sequences of the target DNA, is carried out at a temperature of 60°C for 4 minutes.

[0103] Said steps are repeated for a number of cycles preferably between 19 and 21 times, more preferably 20 cycles.

[0104] Next, a predetermined amount of said reaction solutions contained in the two wells are collected and combined so as to generate a single solution.

[0105] The next step in said amplifying step involves the partial digestion of nucleic acid amplicons.

[0106] The next step in said step of preparing libraries involves the binding of adapters to amplicons by a ligation reaction.

[0107] Following said ligation step, a step e) of purifying the library is carried out by purification with “magnetic beads”. By this method, an amount of solution containing magnetic beads equal to 1.5 times the volume of the solution to be purified is added. After allowing said solution to incubate for 5 minutes at room temperature, the tubes are incubated on a magnet for a time between 5 and 10 minutes. After that, the resulting aqueous phase is removed and two washes of the remaining beads are performed with 70% ethanol. After the second wash, all ethanol is removed and the samples are left for a time between 5 and 10 minutes, preferably 10 minutes, at room temperature to allow any residual ethanol to evaporate. Next, 50 ul of Tris EDTA (TE) is added to allow elution of the DNA thus purified, which will detach from the beads and be transferred to a new tube.

[0108] Step f) of parallel amplicon sequencing is carried out through NGS instrumentations. For example, the step of parallel amplicon sequencing can be carried out by Thermo Fisher Scientific's Gene Studio S5 Prime sequencer. This type of sequencer takes advantage of lonTorrent technology, a parallel DNA sequencing technology based on the detection of hydrogen H ions released during sequencing.

[0109] Indeed, it is well known that this type of sequencing is performed inside a semiconductor microchip that measures the pH change determined by the release of hydrogen ions following the incorporation of nucleotides by the polymerase.

[0110] The amount of protons released is proportional to the number of bases incorporated. Further object of the present invention is a gene (or multi-gene) panel, or a combination of genes, comprising (or alternatively consisting of) the FGFR1, NF1, SDHA, SDHB, SDHC, and SDHD genes.

[0111] In embodiments, the panel comprises (or consists of) nucleotide sequences derived from the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

[0112] In embodiments, the panel comprises the genes (or, alternatively, consists of the genes) set forth in Table 2.

[0113] Advantageously, the gene panel of the invention can be used in the ex vivo diagnosis of gastrointestinal stromal tumors (preferably not caused by mutations in KIT, PDGFR-a, KRAS and BRAF genes), particularly through parallel sequencing (i.e., Next Generation Sequencing) of FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes. Therefore, further object of the present invention is the use of a panel which comprises nucleotide sequences derived from the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, in particular, not caused by mutations in KIT, PDGFR-a, KRAS and BRAF genes, i.e., in the case where KIT, PDGFR-a, KRAS and BRAF genes do not have mutations.

[0114] Advantageously, the use of such multi-gene panel for NGS sequencing allows all possible potentially identifiable alterations in WT GISTs to be simultaneously identified.

[0115] The present invention further relates to a kit for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by mutations in KIT / PDGFRA / KRAS / BRAF genes.

[0116] Specifically, such kit comprises the gene panel according to the invention, that is, a panel for next-generation sequencing (NGS, or parallel sequencing), comprising the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

[0117] In embodiments, said kit comprises reagents adapted to produce a library of amplicons referring to the genomic sequences of the FGFR1, NF1, SD HA, SDHB, SDHC and SDHD genes, and / or means to perform parallel sequencing of the FG'F 7, NF1, SDHA, SDHB, SDHC and SDHD genes.

[0118] In embodiments, the library comprises or, alternatively, is constituted by, the amplicons set forth in Table 1 below.

[0119] EXPERIMENTAL SECTION

[0120] Example 1 - Multi-gene panel for the detection of gastrointestinal stromal tumors GISTs

[0121] DNA isolated and quantified from isolated tissue samples is analyzed by multigenic NGS panel according to the invention.

[0122] The use of the invention's gene panel enables the genomic regions of genes associated with the onset of GIST tumors to be identified.

[0123] The genes under analysis according to the present invention, and their references in scientific databases, are set forth in Table 2 below.

[0124] The multi-gene panel used includes the genes set forth in Table 2 below.

[0125] The use of the multi-gene panel for NGS sequencing comprising the aforementioned genes allows all possible potentially identifiable mutations in WT GISTs to be simultaneously identified. Example 2- Preparation of DNA amplicon library

[0126] The above described steps of preparing the libraries allow a series of DNA amplicons configured to detect target DNA sequences to be obtained.

[0127] Said amplicons are then used to make a genomic panel for NGS sequencing able to simultaneously identify alterations in said genes associated with rare molecular subtypes of GIST tumors.

[0128] The following Tables show the details of the library / gene panel amplicons.

[0129] More specifically, the following details are set forth: the amplicon identifier (ID), the target gene symbol, the reference (NCBI transcript identifier), the version of the human genome, the chromosome harboring the target gene, the start point and end point related to the position of the amplicon sequence on the genome, and the primer pool (related to the regions denoted by the number 1 or 2, respectively) within which that particular amplicon is located.

[0130] Specifically, Table 3 below shows the amplicons for the FGFR1 gene.

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144] H

[0145] Table 7

[0146] Amplicon Gene Reference Genome Cr. Amplicon Amplicon End Primer

[0147] Identifier (ID) Symbol Version Start Pool

[0148] SDHC 1.7954 SDHC NM_003001.5 hgl9 chrl 161284179 161284316 1

[0149] SDHC 2.12855 SDHC NM_003001.5 hgl9 chrl 161293348 161293453 2

[0150] SDHC-3.63994 SDHC NM_003001.5 hgl9 chrl 161298189 161298309 2

[0151] SDHC 3.72133 SDHC NM_003001.5 hgl9 chrl 161298115 161298232 1

[0152] SDHC 4.23659 SDHC NM_003001.5 hgl9 chrl 161310351 161310462 2

[0153] SDHC 5.13284 SDHC NM_003001.5 hgl9 chrl 161326456 161326582 1

[0154] SDHC 5.29529 SDHC NM_003001.5 hgl9 chrl 161326514 161326642 2

[0155] SDHC 6.10463 SDHC NM_003001.5 hgl9 chrl 161332091 161332218 1

[0156] SDHC 6.3442 SDHC NM_003001.5 hgl9 chrl 161332283 161332408 2

[0157] SDHC 1.3056 SDHC NM_003001.5 hgl9 chrl 161293416 161293553 2

[0158] H

[0159] Table 8

[0160] Amplicon Gene Reference Genome Cr. Amplicon Amplicon End Primer

[0161] Identifier (ID) Symbol Version Start Pool

[0162] SDHD 1.67452 SDHD NM_003002.4 hgl9 chrl 1 111957573 111957704 2

[0163] SDHD 2.39414 SDHD NM_003002.4 hgl9 chrl 1 111958562 111958683 2

[0164] SDHD 2.93032 SDHD NM_003002.4 hgl9 chrl 1 111958594 111958720 1

[0165] SDHD 3.31177 SDHD NM_003002.4 hgl9 chrl 1 111959560 111959687 1

[0166] SDHD 3.55684 SDHD NM_003002.4 hgl9 chrl 1 111959656 111959778 2

[0167] SDHD 4.40818 SDHD NM_003002.4 hgl9 chrl 1 111963710 111963840 2

[0168] SDHD 5.32838 SDHD NM_003002.4 hgl9 chrl 1 111965497 111965622 1

[0169] SDHD 5.51815 SDHD NM_003002.4 hgl9 chrl 1 111965599 111965737 2

[0170] SDHD 1.33431 SDHD add NM_003002.4 hgl9 chrl 1 111963840 111963965 2

[0171] Example 3 - Specificity of the diagnostic method for the very rare subtypes of gastrointestinal stromal tumors

[0172] The previously obtained amplicon library (see Example 2) was used for analysis of samples in which KIT / PDGFRA / RAF / RAS genes have at least one mutation. This condition is the most prevalent one since, as mentioned earlier, about 90% of these types of neoplasms are determined by mutations in the sequence of said genes.

[0173] In this case, 10 different samples were analyzed.

[0174] None of these samples showed additional variants by the diagnostic method used. In other words, none of said samples have mutations in FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

[0175] The results obtained show that none of said samples have mutations detectable by the method that is the object of the present invention.

[0176] This demonstrates that this diagnostic method has a high clinical specificity of 100% for the target genes to be analyzed, namely FGFR1, NF1, SDHA, SDHB, SDHC and SDHD.

[0177] Example 4 -Analysis of very rare subtypes of gastrointestinal stromal tumors

[0178] The previously obtained amplicon library (see Example 2) was used to analyze 30 isolated samples associated with gastrointestinal stromal tumor cases negative for variants in KIT / PDGFRa / RAF / RAS.

[0179] All samples used are isolated samples fixed in paraffin and embedded in paraffin (FFPE).

[0180] All samples analyzed were found to be mutated in one of the target genes of the diagnostic method that is the object of the present invention. In these cases, the mutations detected were classified as “Pathogenic” and / or “Likely Pathogenic”.

[0181] This way, clinical framing can be accurately established.

[0182] Only four cases remained clinically "unresolved" (Variants of Uncertain Significance, VUS) after analysis by the diagnostic method that is object of the present invention. Table 9 below shows in detail the type of mutation and the site where it occurred.

[0183]

[0184] The results obtained demonstrate that the diagnostic method of the present invention can detect rare molecular subtypes associated with gastrointestinal stromal tumors GISTs with a high degree of accuracy.

[0185] Furthermore, these results demonstrate the ability of the diagnostic method object of the present invention to simultaneously analyze multiple target genes.

[0186] Example 5 - Kit for ex vivo molecular diagnosis of gastrointestinal stromal tumors GISTs

[0187] As discussed above, the present invention provides a kit for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, particularly those not caused by gene mutations in KIT / PDGFRA / KRAS / BRAF genes.

[0188] This kit comprises the multi-gene next-generation sequencing panel comprising the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

[0189] The sequences of these genes are described in the section "Example 1 - Multi-gene panel for the detection of gastrointestinal stromal tumor GISTs" of the present description.

[0190] Furthermore, this kit comprises reagents adapted to produce amplicons referenced to the genomic sequences of the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes in the gene panel.

[0191] Specifically, the amplicons of said library are set forth in section “Example 2 - Preparation of DNA amplicon library”.

[0192] The amplicons of said library allow target genomic regions of genes in said gene panel to be sequenced and to determine whether there is at least one mutation associated with WT GIST tumors.

Claims

CLAIMS1. A method for ex vivo molecular diagnosis of gastrointestinal stromal tumors, comprising the steps of: a) Providing at least one isolated sample comprising at least one nucleic acid; b) Extracting said nucleic acid; c) Quantifying said previously extracted nucleic acid; d) Preparing at least one amplicon library of said previously extracted nucleic acid, said at least one amplicon library comprising sequences obtained from the genes FGFR1, NFI, SDHA, SDHB, SDHC and SDHD e) Purifying said at least one amplicon library; f) Simultaneous sequencing said amplicons.

2. The method according to claim 1, wherein said gastrointestinal stromal tumors are not due to mutations in at least one gene selected from KIT, PDGFR-a, NRAS, KRAS and BRA I '.

3. The method according to claim 1 or 2, wherein said gastrointestinal stromal tumors are not due to mutations in at least one gene selected from KIT and PDGFR-a.

4. The method according to any one of the preceding claims, wherein said simultaneous sequencing of said amplicon libraries is carried out by Next Generation Sequencing (NGS).

5. The method according to any one of the preceding claims, characterized in that said at least one amplicon library is constituted by sequences obtained from the FGFR1, NFI, SDHA, SDHB, SDHC and SDHD genes only.

6. The method according to any one of the preceding claims, characterized in that said at least one amplicon library is constituted by amplicons as set forth in Table 1.

7. The method according to any one of the preceding claims, characterized in that said at least one nucleic acid is DNA.

8. The method according to any one of the preceding claims, wherein said isolated sample is a tissue sample selected from tissue fixed in formalin and embedded in paraffin (FFPE) or fresh / frozen tissue.

9. The method according to any one of the preceding claims, wherein said isolated sample is fixed in formalin and embedded in paraffin.4910. The method according to any one of the preceding claims, wherein said isolated sample is obtained from an isolated tissue in which mutations in the KIT, PDGFR-a, NRAS, KRAS and BRAF genes are absent.

11. The method according to any one of the preceding claims, characterized in that5 said step d) comprises a first and a second primer, said first and second primers having different nucleotide sequences.

12. A gene panel for ex vivo molecular diagnosis of gastrointestinal stromal tumors, comprising the I'GFR / , NF1, SDH A, SDHB, SDHC and SDHD genes.

13. The gene panel according to claim 12, characterized in that it comprises10 amplicons obtained from the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

14. Use of the gene panel according to claim 12 or 13 in the ex vivo diagnosis of gastrointestinal stromal tumors (GIST), wherein said gastrointestinal stromal tumors are not due to mutations in the KIT, PDGFR-a, NRAS, KRAS and BRAF genes.

15. A kit for the ex vivo molecular diagnosis of gastrointestinal stromal tumors,15 comprising said panel according to claim 12 or 13 and further comprising reagents and / or means adapted to produce a library of amplicons referring to the genomic sequences of the FGFR1, NF1, SD HA, SDHB, SDHC and SDHD genes, and / or to perform parallel sequencing of the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes.

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