ES2244308B1 - METHOD OF MOLECULAR DIAGNOSIS AND TREATMENT IN UNSTABLE SPORTS CANCER. - Google Patents

Abstract

Molecular diagnostic method and treatment in unstable sporadic colorectal cancer. The present invention describes a method of molecular diagnosis and treatment in unstable sporadic colorectal cancer based on the fact that HNPCC tumors associated with MLH1 or MSH2 mutations are all negative for the V599E mutation while this mutation is present in approximately half of sporadic tumors. , allowing to rule out HNPCC cancer associated with MLH1 or MSH2 mutations and avoiding the laborious detection analysis in these genes. The present invention has application in the diagnosis and prevention of sporadic colorectal cancer.

Description

Molecular diagnosis method and treatment in unstable sporadic colorectal cancer.

Object of the invention

The present invention relates to a method of molecular diagnosis between sporadic and familial forms of HPNCC in colorectal cancer patients that can be used as a diagnostic criterion for these tumors as well as in the prevention and treatment thereof.

Background of the invention

In absolute terms, cancer is the second Cause of death in Spain. Among the multiple types of cancer highlights colorectal cancer that caused 11% of deaths for cancer in men and 15% in women according to data from 1999.

The cumulative risk during the life of getting the disease is 5-6%, being influenced by life habits and hereditary factors. The shape The most frequent colorectal cancer is sporadic (80%) existing cases with hereditary components: polyposis Familial adenomatous (FAP) (0.01%) and colorectal cancer hereditary non-polyposic (HNPCC) (5-10%) (Lynch HT, Smyrk TC, Watson P, Lanspa SJ, Lynch JF, Lynch PM, Cavalieri RJ, Boland CR. Genetics, natural history, tumor spectrum, and pathology of hereditary nonpolyposis colorectal cancer: an updated review. Gastroenterology 1993 May; 104 (5): 1535-49. Review), in which the Affected individuals develop in intermediate stages - early your life (before age 50) tumors of the colon, stomach, ovary, endometrium and other organs.

The main genetic factors have been be determined in hereditary syndromes, both of the familial polyposis as in hereditary cancer syndrome non-polyposic colorectal, as well as in colorectal cancer sporadic. Colorectal tumor development is possibly the consequence of a series of molecular events that begin with one or several mutations or epigenetic events and continue with progression phenomena, in which they may be involved both genetic and environmental factors.

Recently, several genetic alterations related to the development of colorectal cancer have been characterized, both in family syndromes and sporadic cancers. Thus, the APC gene has been located on the long arm of chromosome 5 and related to FAP and most sporadic cancers, while, on the other hand, the MLH1 and MSH2 genes have been identified as the main responsible for the HNPCC. On the other hand, activations have also been identified by point mutation of oncogenes such as k-ras or increased expression of the messenger RNA of the c-myc oncogene and specific losses of the suppressor gene material, specifically those located on chromosomes 5 (APC ) and 17 (p53), especially in sporadic cases.

Clinically, the HNPCC is mainly defined by its early onset (<50 a) and by the presence of cancers in first-degree relatives in at least two contiguous generations. The clinical criteria are included in the so-called Amsterdam I, II and Bethesda criteria (Vasen HFA, Mecklin JP, Khan PM, Lynch HT. The international collaborative group on Hereditary Nonpolyposis colorectal cancer (ICG-HNPCC). Dis. Colon Rectum 1991; 34: 424-5; Vasen HF, Watson P, Mecklin JP, Lynch HT. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology. 1999 Jun; 116 (6) : 1453-6; Rodriguez-Bigas MA, Boland CR, Hamilton SR, Henson DE, Jass JR, Khan PM, Lynch H, Perucho M, Smyrk T, Sobin L, Srivastava S. A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines. J Natl Cancer Inst. 1997 Dec 3; 89 (23): 1758-62). The investigations carried out showed that HNPCC tumors were characterized by the presence of somatic alterations in numerous microsatellite sequences, which consisted of repeats of one, two or more nucleotides (Aaltonen LA, Peltomaki P, Leach FS et al . Clues to the pathogenesis of familial colorectal cancer. Science 1993; 260: 812-6.). Other studies showed that a similar subset of sporadic tumors (15%) also showed insertions or deletions in simple repetitive sequences, particularly microsatellites (lonov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic mutations in simple repeated sequences_reveal a new mechanism for colonic carcinogenesis. Nature 1993; 363: 558-61; Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993; 260: 816-9; Aaltonen LA, Peltomaki P, Leach FS et al . Clues to the pathogenesis of familial colorectal cancer. Science 1993; 260: 812-6.). These sporadic tumors showed common characteristics with those that developed in Lynch syndrome or HNPCC. These data suggested that HNPCC tumors and a certain subset of sporadic tumors were associated with a defect that caused DNA replication errors and caused alterations or instability in microsatellites (Ionov Y, Hairstyle MA, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993; 363: 558-61; Aaltonen LA, Peltomaki P, Leach FS et al . Clues to the pathogenesis of familial colorectal cancer. Science 1993; 260: 812- 6.).

It was later observed that the alteration caused by dysfunction of the DNA repair genes (mismatch repair system or MMR) MLH1, MSH2 and MSH6 produced microsatellite instability (MSI-H) in the tumors of patients with Lynch syndrome (Strand M, Prolla TA, Liskay RM, Petes TD. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature. 1993 Sep 16; 365 (6443): 274-6; Peltomaki P, Lothe RA, Aaltonen LA , et al . Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome. Cancer Res 1993; 53: 5853-5; Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. 1993; 260: 816-9). Although 90% of the mutations described in HNPCC so far affect the MLH1 and MSH2 genes, however, these mutations are only present in approximately 50% of cases of HNPCC.

This has made the diagnosis strategies of HNPCC based on both clinical and molecular criteria, with the detection of MSI-H being an important point of inclusion in genetic counseling programs. However, due to cost and time problems, it has not been considered effective to perform MSI-H screening in all colorectal cancer patients. Despite all this, if we exclude early onset and family history, HNPCC and MSI-sporadic tumors are virtually indistinguishable and the distinction between the two goes through the laborious sequencing of the MLH1, MSH2 and MSH6 genes .

Recently, (Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S , Hawes R, Hughes J, Kosmidou V, Menzies A, Mold C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K , Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R , Marshall CJ, Wooster R, Stratton MR, Futreal PA (2002) Mutations of the BRAF gene in human cancer. Nature 417: 949-54) it has been discovered that the BRAF oncogene (B-Raf proto-oncogen serine / threonine-protein quinase (EC
2.7.1.-) (p94) (v-Raf murine sarcoma viral oncogene homolog B1)) is activated by mutation in 70% of melanomas, 10% of colorectal cancers and a minor subset of other tumors. The activated form of BRAF has a preferential point mutation site (hotspot: V599E), represented by a base change at position 1796 of the cDNA: A → T resulting in a substitution of amino acid 599 which becomes glutamic acid in instead of the original valine. This mutation represents 80% of the mutated forms of BRAF and has an increased kinase activity and transforming properties in NIH3T3 cells. Also recently (Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. RAF / RAS oncogenes and mismatch-repair status. Nature 2002; 418: 934) an association between V599E and colon tumors has been demonstrated with repair deficit, that is, unstable for microsatellites (MSI-H). However, it has been reported that mutations of BRAF in colon cancer are not attributable to defects in the MMR system (Wang L, Cunningham JM, Winters JL, et al. BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair Cancer Res 2003; 63: 5209-12), so its involvement in the HNPCC is a question and is yet to be determined.

Therefore, there is a need in the state of the art of identifying new genes and / or responsible mutations and / or indicators of HNPCC cancers versus MSI-sporadic.

The identification of this / these genes and their possible mutations would allow a selective molecular diagnosis and rapid between sporadic MSI cancer and HNPCC cancer that would allow include it in normal clinical practice and perform a typing Universal molecular of colorectal tumors diagnosed.

At the same time it would produce cost savings toilets and shorter wait times by limiting the sequencing of the genes responsible for MMR to those cases negative

It would also allow the adoption of new prognostic criteria and therapeutic guidance in cases positive as it would allow adapting current therapies and customize them to those cases in which the hereditary character It is indicated early.

Finally it would allow the design of therapies selective aimed at inactivating these genes.

Description of the invention

The present invention aims at a method of diagnosing colorectal cancer which comprises analyzing a sample to determine the presence of a point mutation V599E in the BRAF gene whose presence allows to rule out the hereditary nature of colorectal cancer as associated hereditary non-polyposic cancer (HNPCC) to mutations of MLH1 or MSH2 or other DNA repair genes and identify the tumor molecularly as sporadic MSI-H.

In particular, said sample can be DNA or RNA and can be isolated from tumor cells obtained by biopsy or any other extraction method. Optionally, the sample analyzed can be the protein encoded by said gene or fragments of it.

The diagnostic method of this invention further comprises a detection by a amplification by PCR, SDA or any other amplification method of DNA. In particular, said amplification is performed by use of allele-specific primers.

Optionally, the detection can be carried out by DNA sequencing, by DNA biochips made with oligonucleotides deposited by any mechanism, by DNA biochips made with oligonucleotides synthesized in situ by photolithography or by any other mechanism, by conformational analysis of polymorphism ( SSCP) or other methods based on conformational analysis or by enzymatic restriction analysis.

The present invention also aims at provide a DNA molecule that when found mutated from specific form allows to classify the tumor as sporadic cancer MSI-H, ruling out hereditary syndrome of HNPCC non-polyposic colorectal cancer or Lynch syndrome.

The present invention also aims to provide a method of prognostic analysis in the evolution of sporadic unstable colorectal cancer differentiated from the rest of sporadic colorectal cancers based on the determination of the activity of the V599E form of the BRAF gene in MSI- colorectal tumors. H. The diagnostic method of the present invention also aims at establishing a personalized therapeutic strategy for unstable sporadic colorectal cancer.

On the other hand, the present invention also aims to provide a method of compound analysis to identify therapeutic agents based on the determination of the activity of the V599E form of the BRAF gene in MSI-H colorectal tumors.

The use of compounds to eliminate the activity of the V599E form of BRAF in sporadic colorectal tumor to stop or reverse the progression of such tumors is also an object of the present invention.

A further objective of the present invention is to provide a pharmaceutical compound based on RNA interference to inhibit the activity of the V599E form of the BRAF gene in MSI-H colorectal tumors.

Another additional object of the present invention is to provide a pharmaceutical compound based on anti-sense oligonucleotides to inhibit the activity of the V599E form of the BRAF gene in MSI-H colorectal tumors.

A subject of the present invention is also to provide a compound based on interfering with the replication or translation of the V599E form of the BRAF gene in MSI-H colorectal tumors.

Likewise, the present invention aims to provide a compound based on compounds that selectively inhibit the serine-threonine kinase activity of the V599E BRAF form in sporadic MSI-H colorectal tumors.

The present invention is based on the fact that a mutated form of the human BRAF oncogene is always absent from colorectal tumors that develop in Lynch Syndrome (HNPCC). This mutated form in nucleotide 1796 A → T implies a change of amino acid from valine to a glutamic and involves the constitutive activation of the Serine-Threonine kinase activity of BRAF . This mutation has been described previously as extensively present in melanomas, some colorectal cancers and other tumors being a preferential mutation (hotspot) that represents up to 92% of substitution mutations in this gene.

At the same time, the available data demonstrate that this V599E mutation is present in, about half of sporadic colorectal tumors presenting high microsatellite instability (MSI-H).

Sporadic colorectal tumors and HNPCC type hereditary are practically indistinguishable histologically and pathologically. HNPCC syndrome is manifested by the appearance of tumors at an early age, the existence of at least two first-degree relatives who have developed tumors and the presence of high microsatellite instability or MSI-H. Despite this, the data clinical-family members are not always clear since about 20% of families with germline mutations do not They meet the Amsterdam criteria. The molecular distinction is very complex, especially because there is also a subset Significant sporadic tumors with MSI-H (approximately 15%).

Therefore, the present invention demonstrates a total dissociation of the HNPCC tumors and the V599E mutation of BRAF , which is present in half of the sporadic colorectal tumors that exhibit high microsatellite instability (MSI-H), which means having at one time first of a molecular tool thanks to which small genotypic changes allow to clearly distinguish both types of tumors.

Currently, patients with Amsterdam criteria are first analyzed for MSI-H and, in the case of giving a positive result, they are subsequently analyzed for MLH-1 and MSH-2 and, if these negative results, analyzed for MSH-6 .

According to the diagnostic method of the present invention guidelines for colorectal tumor analysis not selected would be as follows:

one

In this way, a patient who has a colorectal tumor diagnosed by a biopsy and with a non-definitive clinical-family assessment would be subjected to preliminary MSI-H tests. If positive, the result is PCR amplification or similar using the following specific primers for exon 15 of the BRAF gene on genomic DNA:

Direct primer 5'-TCATAATGCTTGCTCTGATAGGA-3 '

Reverse primer 5'-GGCCAAAAATTTAATCAGTGGA-3 '

Also contemplated as part of the present invention is the use of an antineoplastic therapy directed specifically at patients with unstable sporadic colorectal cancer showing the activated form V599E BRAF . This therapy combines any standard therapeutic protocol to which the patient may end up developing resistance together with a specific anti-BRAF therapy. Examples of standard antitumor therapies are hormonal treatments, with ionizing radiation, with cytotoxic and / or cytostatic drugs, with antibodies etc ,. Anti-BRAF therapy begins by determining the presence of the V599E mutation in the tumor mass or other body sample. The inactivation of the mitogenic pathway activated by V599E BRAF may involve the containment and / or regression of the tumor mass. The oncogenesis process occurs through the acquisition and selection of multiple somatic mutations each of which contributes to the survival and expansion of the tumor. The malignant behavior of the tumor can be mitigated or reversed by the selective decrease of said oncogenes. Chemical or antibody inhibition approaches have been tested with some success with beta-catenin in colorectal cancer and with BCR-ABL in chronic myeloid leukemia. Thus, these observations suggest that certain oncogenes could be susceptible to a disruption causing the tumoral phenotype to fall or allowing the efficacy of the combined standard treatment. Among the mechanisms of gene disruption available, interference by small RNAs (RNAi) is one of the most promising approaches, initially discovered in petunias (Napoli C, Lemieux C, and Jorgensen R. (1990) Introduction of a chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279-289) its molecular mechanism of action has been described (Hammond SM, Caudy AA, Hannon GJ. (2001) Post-transcriptional Gene Silencing by Double-stranded RNA, Nature Rev Gen 2: 110-119) and has raised great expectations in its antitumor applications (Kittler R, Buchholz F. RNA interference: gene silencing in the fast lane. Semin Cancer Biol. 2003 Aug; 13 ( 4): 259-65; Deveraux QL, Aza-Blanc P, Wagner KW, Bauerschlag D, Cooke MP, Hampton GM. Exposing oncogenic dependencies for cancer drug target discovery and validation using RNAi. Semin Cancer Biol. 2003 Aug; 13 (4 ): 293-300; Bedford JS, Liber HL Applications of R NA interference for studies in DNA damage processing, genome stability, mutagenesis, and cancer. Semin Cancer Biol. 2003 Aug; 13 (4): 301-8).

An example of this effect can be postulated with cell lines of colorectal tumor origin that show a positive K-RAS negative BRAF genotype. An example of these lines is the Line

Linia RKO, with clonogenic capacity. Ref. J Biol Chem. 2002 May 24; 277 (21): 19156-65.

Linia HT29, with clonogenic capacity .: Ref. Br J Cancer. 2003 Dec 15; 89 (12): 2277-83.

For a specific treatment with RNA from small interference (21-mers) you have to select specific sequences within the cDNA for use as anti-BRAF target. The candidate sequences are compared using BLAST or similar algorithms to eliminate those that are not unique. In the following table they are described as example some of the possible targets in the cDNA

2

Tumor cells in culture are known for their loss of contact inhibition which makes them grow to confluence in a crop and subsequently form aggregations that continue to grow in three dimensions (foci) These foci mimic somehow tumor growth. In a type experiment the RKO or HT29 cells grown under standard conditions in the middle of Dulbecco Eagle modification (DMEM) supplemented with 10% fetal calf serum (FCS), 100 units / ml of penicillin, and 100 µg / ml of Streptomycin, under 5% CO2 form foci. On the contrary, the RNAi treated cells do not show an ability to form these foci and show a growth rate different from those untreated cells.

Also contemplated as part of the present invention is the use of the activated form V599E BRAF to perform analyzes of therapeutic compounds and / or pharmaceutical compositions that can remedy the oncogenic potential of BRAF in unstable sporadic colorectal cancer. These therapeutic compounds and / or pharmaceutical compositions can bind to the mutated BRAF protein and restore its normal activity. Alternatively, these therapeutic compounds and / or pharmaceutical compositions can modify the expression of alternative cell signaling pathways that can compensate for uncontrolled activation of BRAF. The analyzes of therapeutic compounds and / or pharmaceutical compositions can be performed comparatively by analysis of direct application to normal cells and cells with the activated version of the oncogene. These cells can be a cell line from a colorectal tumor or other type of tumor and with a genotype similar to that found in excised tumors (for example K-Ras- / BRAF +) and that can be a reasonable biological model for study of tumor progression / regression. Alternatively these cells can be obtained directly from tumors removed and placed in culture.

Brief description of the figures

Fiqura 1A. Agarose gel in ethidium bromide of the BRAF PCR products subsequently used in the SSCP gels.

M: molecular weight marker. Sizes indicated On the side.

C-: negative control without mutation in BRAF (line tumor cell).

C +: positive control with the V599E mutation (tumor cell line). The arrow indicates the expected size for BRAF.

Examples of HNPCC cases are shown associated with MLH1 (lanes 4 to 9) and HNPCC cases associated with MSH2 (lanes 10 to 15). It is observed that the size of the fragments of PCR corresponds to that expected according to BRAF primers used for amplification.

Fiqura 1B. Polymorphism detection single chain conformational (SSCP) of HNPCC cases with germline mutations of MLH1 and MSH2 analyzed by mobility by a non-denaturing vertical radioactive gel. In this Figure shows the amplicons obtained from tumors HNPCC colorectals associated with MLH1 (lanes 4-9) and to MSH2 (lanes 10-15) along with the controls positive (C +) and negative (C-). The arrow indicates the bands altered due to BRAF-V599E.

Description of a preferred embodiment

An embodiment is described below. preferred, but not limited to, the invention.

Amplification of DNA samples obtained from tumors colorectals previously classified as HNPCC

The amplification reaction of DNA samples obtained from colorectal tumors previously classified as HNPCC was carried out on genomic DNA using specific primers for exon 15 of the BRAF gene, such as for example the direct primer identified as SEQ. ID 1 and the reverse primer identified as SEQ. ID 2, starting at about 50 ng of mold and a cycle with a denaturation phase of 30 s. to 94 ° C followed by banding for 20 s. at 60 ° C and a subsequent extension of 30 s. at 72 ° C. This cycle was repeated during 30 or 35 times As seen in Figure 1A, the fragments amplified had the expected size according to the primers used. Subsequently the PCR product was purified by means of a purification kit of amplification products Wizard? PCR Preps DNA Purification System from Promega and subsequently it was sequenced following the instructions of the ABI kit Prism Dye terminator by Perkin Elmer.

A similar sequence of steps can be performed from RNA prior to the construction step of a 1st complementary cDNA chain using a mixture of hexamer-random primers that statistically prioritize all messengers in the cell and adding the mononucleotides and a buffer necessary for the action of the enzyme Reverse Transcriptase. From the construction of the first complementary DNA chain, a BRAF cDNA fragment containing the preferential mutation zone can be amplified using in this case oligonucleotide primers adapted to the template without introns.

Single chain conformational polymorphism detection (SSCP) of the different colorectal tumors previously classified as HNPCC

The amplification product analysis obtained can be carried out by direct sequencing or by SSCP and sequencing of the amplicons with polymorphisms. When they are present the polymorphisms cause a conformational change that it results in a different migration speed. In figure 1B you show the amplicons that were obtained from tumors HNPCC colorectals associated with MLH1 and MSH2 and analyzed by mobility through a non-denaturing vertical radioactive gel. As can be seen, all cases were negative for V599E

BRAF was amplified by PCR using the same protocol mentioned above, but adding radioactive isotopes, specifically alpha-32 P dCTP. The reaction product was resolved after denaturation at 95 ° C, in a 5% non-denaturing polyacrylamide gel in the mutation detection enhancement gels system (MDE-Flowgen, Rockland, ME, USA), at 8 ° C for 16 hours. It was dried at 80 ° C for 1 hour and its autoradiographic exposure and later developed, at 24 hours. The altered PCR bands were subsequently purified and sequenced in an ABI Prism 377 automatic sequencer (Perkin-Elmer, Foster City, CA, USA) using ABI Prism Dye Terminator Cycle Sequencing Kit (Perkin-Elmer).

Claims (13)

1. Molecular analysis method to rule out the hereditary nature of colorectal cancer with microsatellite instability, characterized in that it comprises analyzing a sample to determine the presence of a point T1796A mutation in the BRAF gene that results in the V599E mutation in the BRAF protein whose presence it allows to rule out the hereditary nature of colorectal cancer as hereditary nonpolyposic cancer (HNPCC) associated with mutations of MLH1 or MSH2 or other DNA repair genes and identify the tumor molecularly as sporadic MSI-H. 2. Molecular analysis method according to claim 1, characterized in that the sample is isolated from tumor cells obtained by biopsy or any other extraction method. 3. Molecular analysis method according to any of claims 1 to 2, characterized in that the sample to be analyzed is DNA. 4. Molecular analysis method according to any one of claims 1 to 2, characterized in that the sample to be analyzed is RNA. 5. Molecular analysis method according to any of claims 1 to 4, characterized in that a detection is performed by amplification by PCR, SDA or any other method of DNA amplification. 6. Molecular analysis method according to claim 5, characterized in that the amplification is carried out using allele-specific primers. 7. Molecular analysis method according to any one of claims 1 to 4, characterized in that a detection by DNA sequencing is carried out. 8. Molecular analysis method according to any one of claims 1 to 4, characterized in that a detection is carried out by DNA biochips made with oligonucleotides deposited by any mechanism. 9. Molecular analysis method according to any one of claims 1 to 4, characterized in that DNA biochips are detected by oligonucleotides synthesized in situ by photolithography or by any other mechanism. 10. Molecular analysis method according to any of claims 1 to 4, characterized in that a detection is carried out by conformational analysis of polymorphism (SSCP) or other methods based on conformational analysis. 11. Molecular analysis method according to any of claims 1 to 4, characterized in that a detection is carried out by enzymatic restriction analysis. 12. Molecular analysis method according to any one of claims 1 to 2, characterized in that the analyzed sample is the protein encoded by said gene or fragments thereof. 13. Method of analysis of compounds with therapeutic potential in colon cancer comprising determine the ability of said compounds to decrease the kinase activity of the mutated form V599E of the BRAF protein that is increased in sporadic colorectal tumor with relation to the activity of the non-mutated protein.