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EP3058103A2 - Détection et surveillance de mutations d'histiocytose - Google Patents

Détection et surveillance de mutations d'histiocytose

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Publication number
EP3058103A2
EP3058103A2 EP14853756.6A EP14853756A EP3058103A2 EP 3058103 A2 EP3058103 A2 EP 3058103A2 EP 14853756 A EP14853756 A EP 14853756A EP 3058103 A2 EP3058103 A2 EP 3058103A2
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EP
European Patent Office
Prior art keywords
mutation
braf
histiocytosis
patient
pcr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14853756.6A
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German (de)
English (en)
Other versions
EP3058103A4 (fr
Inventor
Filip JANKU
Mark G. Erlander
Cecile Rose VIBAT
Karena Kosco
Omar Abdel-Wahab
Eli L. DIAMOND
David M. HYMAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cardiff Oncology Inc
Original Assignee
Trovagene Inc
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Filing date
Publication date
Application filed by Trovagene Inc filed Critical Trovagene Inc
Publication of EP3058103A2 publication Critical patent/EP3058103A2/fr
Publication of EP3058103A4 publication Critical patent/EP3058103A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This present invention is directed to methods of determining treatment options, and methods of treating, histiocytosis. More specifically, the invention provides improved assays for detecting and quantitating mutations associated with histiocytosis, and the determination of treatment options that utilize the results of those assays.
  • Histiocytosis is a group of rare diseases characterized by the proliferation of histiocytes, or cells derived from monocytes, e.g., tissue macrophages and dendritic cells. Three groups of histiocytosis are recognized. One group is macrophage disorders, including hemophagocytic lymphohistiocytosis and Rosai-Dorfman Disease. The second group is malignant histiocytosis, and the third group is dendritic cell disorders, including Langerhans cell histiocytosis (LCH), juvenile xanthogranuloma, and Erdheim-Chester disease (ECD).
  • LCH Langerhans cell histiocytosis
  • ECD Erdheim-Chester disease
  • ECD is a rare form of non- Langerhans cell histiocytosis affecting adults, which is associated with xanthogranulomatous infiltration of foamy macrophages (Janku et al., 2010, 2013; Arnaud et al., 2011)
  • the V600E mutation in BRAF and other mutations in the RAS-RAF-MEK-ERK and RAS- PI3K-AKT signaling pathways are associated with various histiocytoses (Badalian-Very et al., 2010; Emile et al., 2013, 2014; Brown et al., 2014; Chakraborty et al., 2014; Arceci, 2014); the V600E BRAF mutation is present in as many as 40-60% of patients with systemic histiocytosis, such as Langerhans Cell Histiocytosis (LCH).
  • LCH Langerhans Cell Histiocytosis
  • the BRAF protein is part of the RAS- RAF-MEK-MAPK signaling pathway that plays a major role in regulating cell survival, proliferation and differentiation (Keshet and Seger, 2010).
  • BRAF mutations constitutively activate the MEK-ERK pathway, leading to enhanced cell proliferation, survival and ultimately, neoplastic transformation (Wellbrock and Hurlstone, 2010; Niault and Baccarini, 2010).
  • all BRAF mutated LCH cases carried the V600E phospho-mimetic substitution which occurs within the BRAF activation segment and markedly enhances its kinase activity in a constitutive manner (Wan et al., 2004).
  • the present invention is based on the discovery that gene mutations associated with histiocytosis are present in cell-free DNA in bodily fluids, and that the presence of those gene mutations can be monitored over time to follow the course of the disease.
  • a method of detecting a mutation in a histiocytosis patient comprises (a) obtaining a sample of a bodily fluid from the patient; and (b) testing the sample for the presence of a mutation in a gene in the RAS-RAF-MEK-ERK or the RAS-PI3K-AKT pathway in cell free DNA (cfDNA) in the bodily fluid.
  • a method of monitoring disease course of a histiocytosis in a patient having a mutation in a gene in the RAS-RAF-MEK-ERK or the RAS-PI3K-AKT pathway comprises
  • the method comprises detecting a mutation in the patient or monitoring disease course of the patient's histiocytosis by the above methods; and selecting and/or applying a treatment or therapy based on the detecting or monitoring.
  • the method comprises
  • FIG. 1 illustrates an exemplary two-step assay design for a 28-30 bp footprint in the target gene sequence.
  • FIG. 2 are graphs of experimental results showing positive and negative controls for the identification of a BRAF V600E mutation.
  • FIG. 3 is a graph showing results from an ECD afflicted patient during treatment with vemurafenib. Sensitivity to the therapy is observed.
  • FIG. 4 is a graph showing results from an ECD afflicted patient treated with anakinra (trade name Kineret ® ) followed by termination of treatment and then administration of treatment with vemurafenib.
  • anakinra trade name Kineret ®
  • FIG. 5 is a graph showing results from an ECD afflicted patient during treatment with anakinra. Sensitivity to the therapy is observed.
  • FIG. 6 is graphs showing that BRAF sampling in urinary cfDNA detection of ECD improves chances of success over biopsies due to the high correlation in performance between urinary cfDNA and both tissue samples and plasma from blood samples.
  • the remainder of FIG. 6 shows a correlation between urinary cfDNA and plasma cfDNA in detecting BRAF wildtype, mutant, and unknown genotypes when using the methods disclosed herein.
  • FIG. 7 is graphs showing BRAF V600E mutant allele burden in cell-free DNA (cfDNA) of urine and plasma from treatment naive patients based on BRAF V600E tissue genotype result.
  • Panel A shows the ratio of BRAF Y600E:BRAF wildtype in urinary cfDNA of patients based on BRAF mutational status of histiocytosis tissue biopsy (BRAF V600E mutant, BRAF wildtype, or BRAF mutational status unknown).
  • Panel B shows the ratio of BRAF Y600E:BRAF wildtype in plasma cell-free DNA of patients based on BRAF mutational status of histiocytosis tissue biopsy. Each point represents a single test result from evaluation before initiation of any therapy. The dashed line indicates the cutpoint indicating the presence of the BRAF V600E mutation.
  • FIG. 8 is graphs showing BRAF V600E mutant allele burden in cell-free DNA (cfDNA) of urine and plasma based on BRAF V600E tissue genotype result.
  • Panel A is pie chart representations of BRAF V600E mutational genotypes as determined by initial tissue biopsy (left) or urinary cfDNA analysis (right). Results were recorded as BRAF V600E mutant (light shade), BRAF V600E wildtype (white), or result indeterminate (dark shade).
  • Panel B shows the ratio of BRAF Y600E:BRAF wildtype in urinary cfDNA of patients based on BRAF mutational status as determined from tissue biopsy (BRAF V600E mutant, BRAF wildtype, or BRAF mutational status unknown).
  • Panel C shows the ratio of BRAF Y600E:BRAF wildtype in plasma cfDNA of patients based on BRAF mutational status as determined from tissue biopsy. Each point represents a single test result from the initial assessment of BRAF Y600E:BRAF wildtype allelic ratio in cfDNA. Dotted points represent samples collected during RAF inhibitor therapy. The dashed line indicates the cutpoint indicating the presence of the BRAFV600E mutation.
  • FIG. 9 is graphs showing the effect of therapy on BRAF V600E mutant allele burden in cell-free DNA (cfDNA) of systemic histiocytosis patients.
  • Panel A shows a comparison of BRAF V600E allele burden in treatment naive urine samples compared with urinary samples acquired anytime during therapy.
  • Panel B shows the effect of RAF inhibitors on cfDNA BRAF V600E mutant allele burden in 7 consecutive patients treated with RAF inhibitors. The initial sample in each patient is prior to initiation of therapy. The dashed line indicates the cutpoint indicating the presence of the BRAFV600E mutation.
  • FIG. 10 shows graphs and clinical imaging results demonstrating dynamic monitoring of serial urinary cell-free DNA (cfDNA) BRAF V600E mutant allele burden in systemic histiocytosis patients.
  • Panel A shows gadolinium-enhanced Tl MRI images of ECD involvement of brain (arrows), and 18 F-FDG-PET images of disease in the right atrium (asterisk) and testes (asterisk), pre-dabrafenib and after 2 months of dabrafenib.
  • Panel B shows urinary BRAF V600E cfDNA results throughout this same patient' s therapy.
  • Panel C shows urinary BRAF V600E cfDNA results of an ECD patient treated with anakinra followed by a period of treatment cessation and then initiation of vemurafenib.
  • Panel D shows maximal intensity projection (MIP) images of 18 F-FDG- PET scan images demonstrating tibial infiltration by ECD pre- vemurafenib, following 10 weeks of vemurafenib, and then 16 weeks after vemurafenib discontinuation in an ECD patient (top) with accompanying urinary cfDNA results for each time point (below).
  • MIP maximal intensity projection
  • FIG. 11 shows radiographic, histologic, and molecular evaluation of KRAS G12S mutant patient with Erdheim-Chester Disease (ECD).
  • Panels A and B show an 18 F-FDG-PET scan result of an ECD patient who was BRAF V600E wildtype by tissue biopsy and urinary and plasma cell- free DNA analysis revealing PET avidity of heart (Panel A) and right atrium specifically (Panel B).
  • Panel C shows a hematoxylin-eosin stained histological section of cardiac tissue biopsy showing a prominent histiocytic infiltrate. Histiocytes have abundant pale staining, finely granular cytoplasm.
  • Panel D shows a screen shot using Integrated Genomics Viewer (IGV) demonstrates the presence of KRAS G12S mutation in DNA from a histiocyte tissue biopsy.
  • Panel E shows next- generation sequencing of PCR enriched amplicon from urine and plasma derived cfDNA confirming KRAS G12S mutation.
  • IGF Integrated Genomics Viewer
  • sample refers to anything which may contain an analyte for which an analyte assay is desired.
  • the analyte is a cf nucleic acid molecule, such as a DNA or cDNA molecule encoding all or part of BRAF.
  • the sample may be a biological sample, such as a biological fluid or a biological tissue.
  • biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid or the like.
  • Biological tissues are aggregate of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).
  • a "patient” includes a mammal.
  • the mammal can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or a pig. In many cases, the mammal is a human being.
  • the present invention is based in part on the discovery that gene mutations associated with histiocytosis, such as BRAF V600E, is present in cell-free DNA in bodily fluids, and that the presence of those gene mutations can be monitored over time to follow the course of the disease. See Examples below.
  • cfDNA Cell-free DNA
  • Detecting and quantifying the amount of mutant cfDNA fragments harboring specific mutations can be used as an alternative to tissue testing.
  • a method of detecting a mutation in a histiocytosis patient comprises (a) obtaining a sample of a bodily fluid from the patient; and (b) testing the sample for the presence of a mutation in cell free DNA (cfDNA) in the bodily fluid.
  • the mutation is in a gene in the RAS-RAF-MEK-ERK or the RAS-PI3K-AKT pathway.
  • Any bodily fluid that would be expected to have cfDNA can be utilized in these methods.
  • Non-limiting examples of a bodily fluid include, but are not limited to, peripheral blood, serum, plasma, urine, lymph fluid, amniotic fluid, and cerebrospinal fluid.
  • the bodily fluid is serum, plasma or urine.
  • any mutation particularly any mutation in a gene in the RAS-RAF- MEK-ERK or the RAS-PI3K-AKT pathway, that leads to enhanced cell proliferation could be utilized in any of the methods disclosed herein.
  • genes in these pathways having mutations associated with histiocytosis are BRAF, PIK3A, NRAS, MAPK1 , ARAF or ERBB3 genes.
  • the mutation is in a BRAF gene.
  • the BRAF mutation is a BRAF V600E mutation, in which a glutamic acid (Glu or E) is substituted for a Valine (Val or V) residue at position or amino acid residue 600 of SEQ ID NO: 9.
  • the BRAF mutation is a substitution of an adenine (A) for a thymine (T) nucleotide at position 1860 of SEQ ID NO: 1.
  • Wildtype Homo sapiens v-raf murine sarcoma viral oncogene homolog Bl, BRAF is encoded by the following mRNA sequence (NM_004333, SEQ ID NO: l) (wherein coding sequence is bolded and the coding sequence for amino acid residue 600 is underlined and enlarged):
  • any of the methods described herein can be utilized with patients having any histiocytosis that is associated with a mutation, since any mutation associated with a histiocytosis would be expected to be represented in bodily fluids and detectable by the methods described herein, as exemplified in the Examples with the BRAF and KRAS mutations.
  • the mutation is in a gene in the RAS-RAF-MEK-ERK or the RAS-PI3K-AKT pathway.
  • the mutation is a KRAS mutation, e.g., a G12A, G12C, G12D, G12R, G12S, G12V or G13D mutation. See Example 7.
  • the patients are humans.
  • the patients may be of any age, including, but not limited to infants, toddlers, children, minors, adults, seniors, and elderly individuals.
  • the histiocytosis is Langerhans Cell Histiocytosis (LCH).
  • the histiocytosis is non-Langerhans Cell Histiocytosis (nLCH).
  • the nLCH is Erdheim-Chester Disease (ECD).
  • an nLCH include benign cephalic histiocytosis, generalized eruptive histiocytoma, (giant cell) reticulohistiocytoma, hemophagocytic lymphohistiocytosis (HLH), indeterminate cell histiocytosis, juvenile xanthogranuloma (JXG), Kikuchi disease, multicentric reticulohistiocytosis, necrobiotic xanthogranuloma, Niemann-Pick disease, progressive nodular histiocytoma, Rosai-Dorfman disease, Sea-blue histiocytosis, and xanthoma disseminatum.
  • Other possible examples are interdigitating dendritic sarcoma and histiocytic sarcoma.
  • the histiocytosis can be cancerous or noncancerous.
  • LCH Langerhans Cell Histiocytosis
  • the mutation can be determined, or quantified, by any method known in the art.
  • Nonlimiting examples include MALDI-TOF, HR-melting, di-deoxy- sequencing, single-molecule sequencing, use of probes, pyrosequencing, second generation high- throughput sequencing, SSCP, RFLP, dHPLC, CCM, or methods utilizing the polymerase chain reaction (PCR), e.g., digital PCR, quantitative-PCR, or allele- specific PCR (where the primer or probe is complementary to the variable gene sequence).
  • the PCR is droplet digital PCR, e.g., as described in the Examples.
  • the mutation is quantified along with the wildtype sequence, to determine the percentage of mutated sequence. In other methods, only the mutation is quantified.
  • the nucleic acids are cf DNA ("cfDNA").
  • the amplified or detected DNA molecule is genomic DNA.
  • the amplified or detected molecule is a cDNA.
  • the nucleic acids is cfRNA or cf mRNA.
  • the assay may be utilized in quantitative, semi-quantitative, or qualitative modes to monitor molecular changes over time.
  • the method is performed quantitatively, such that the amount of the gene alteration is quantitatively determined and may be quantitatively compared to another measurement.
  • methods for quantitative determinations include quantitative PCR or sequencing.
  • the method is performed semi-quantitatively, such that the amount of the gene alteration may be determined and then compared to another measurement simply to determine a relative increase or decrease relative to each other.
  • the method is performed qualitatively, such that the mutation is determined as detectable or not detectable.
  • the detection limits for the presence of a gene alteration (mutation) in cf nucleic acids may be determined by assessing data from one or more of negative controls (e.g. from healthy control subjects or verified cell lines) and a plurality of patient samples.
  • the limits may be determined based in part on minimizing the percentage of false negatives as being more important than minimizing false positives.
  • One set of non-limiting thresholds for BRAF V600E is defined as less than about 0.05% of the mutation in a sample of cf nucleic acids for a determination of no mutant present or wild-type only; the range of about 0.05% to about 0.107% as "borderline", and greater than about 0.107% as detected mutation.
  • a no-detection designation threshold for the mutation is set at less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, or less than about 1% detection of the mutation relative to a corresponding wildtype sequence.
  • a no-detection designation threshold for the mutation is set at less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, or less than about 1% detection of the mutation relative to a corresponding wildtype sequence.
  • the patient has not previously undergone testing for a mutation in a gene, e.g., in the RAS-RAF-MEK-ERK or the RAS-PI3K-AKT pathway.
  • the method are used to determine whether a specific mutation is involved in the histiocytosis, and whether a medicament that targets the product of the gene having the mutation could be effective.
  • a BRAF V600E mutation the patient might be treated with a BRAF inhibitor such as vemurafenib, sorafenib or dabrafenib.
  • akinra (Kineret ® ), a recombinant form of interleukin-1 receptor antagonist (RA), anthracyclines, cladribine, etoposide, vinblastine, alkylating agents, antimetabolites, vinca alkaloids, immunotherapy (alpha interferon), systemic corticosteroids, immunosuppressants, methotrexate, tamoxifen, imatinib (Gleevec ® ), infliximab, tocilumab (Actemra ® ), surgical removal or reduction of any mass which has formed, radiation treatment, antibiotics, and modafinil (Provigil ® ) and other chemotherapy.
  • RA interleukin-1 receptor antagonist
  • anthracyclines cladribine
  • etoposide etoposide
  • vinblastine alkylating agents
  • antimetabolites vinca alkaloids
  • immunotherapy alpha interferon
  • systemic corticosteroids
  • the patient has been previously tested and a mutation determined, and the subsequent tests are to evaluate the course of the disease and/or the effectiveness of treatment.
  • the detecting may identify the non-responsiveness to a treatment or therapy, and the selecting and/or applying comprises a different treatment or therapy. In other cases, the detecting may identify the responsiveness to a treatment or therapy, and the selecting and/or applying comprises continuation of the same treatment or therapy.
  • the present invention is also directed to a method of monitoring disease course of a histiocytosis in a patient having a mutation in a gene in the RAS-RAF-MEK-ERK or the RAS- PI3K-AKT pathway is provided.
  • the method comprises
  • the monitoring of the mutation is accompanied by a determining the disease burden, e.g., by radiography, computed tomography (CT) scanning, positron emission tomography (PET), or PET/CT scanning, and comparing the determined amount of mutation to the disease burden. This is useful to determine whether, or confirm that the mutation being monitored is actually the driver of the disease.
  • CT computed tomography
  • PET positron emission tomography
  • PET/CT scanning PET/CT scanning
  • the determined amount of mutation is not compared to disease burden, either at one, more than one, or all the mutation monitoring times. Given the reliability of the mutation monitoring procedures described herein, a disease burden assessment need not be made at each time point, thus saving the patient a disease burden assessment.
  • these methods may be used to confirm the maintenance of a disclosed treatment or therapy against histiocytosis, or to change the treatment or therapy against the disease.
  • the disclosure includes increasing the treatment or therapy; reducing the treatment or therapy, optionally to the point of terminating the treatment or therapy; terminating the treatment or therapy with the start of another treatment or therapy; and adjusting the treatment or therapy as non-limiting examples.
  • Non-limiting examples of adjusting the treatment or therapy include reducing or increasing the therapy, optionally in combination with one or more additional treatments or therapies; or maintaining the treatment or therapy while adding one or more additional treatments or therapies.
  • the observation of cell-free (cf) nucleic acids identifies an increase in the levels of cf nucleic acids containing the mutation following the start of a treatment or therapy. Following the increase, the observation may reach an inflection point, where the levels decrease, or continue to increase. The presence of an inflection point may be used to determine responsiveness to the treatment or therapy, which may be maintained or reduced. A continuing decrease in the levels to be the same as, or lower than, the levels before the start of treatment of therapy is a further confirmation of responsiveness.
  • the absence of an inflection point indicates resistance to the treatment or therapy and so may be followed by terminating administration of the treatment or therapy, or administering at least one additional treatment or therapy against the disease or disorder to the patient, reducing the treatment of the subject with the treatment or therapy and administering at least one additional treatment or therapy against the disease or disorder to the subject.
  • an additional inflection point may be observed. This may indicate the development of resistance to the treatment or therapy and be followed by terminating administration of the treatment or therapy, or administering at least one additional treatment or therapy against the disease or disorder to the subject, or reducing the treatment of the subject with the therapy and administering at least one additional therapy against the disease or disorder to the subject.
  • the samples can be tissue samples or bodily fluid samples. Any tissue sample that provides sufficient nucleic acids to determine the presence of the mutation may be utilized.
  • the tissue sample is from abnormal tissue associated with the histiocytosis, such as from a lesion or tumor.
  • the tissue can be fresh, freshly frozen, or fixed, such as formalin-fixed paraffin-embedded (FFPE) tissues.
  • FFPE formalin-fixed paraffin-embedded
  • the sample can be obtained by any means, for example via a surgical procedure, such as a biopsy, or by a less invasive method, including, but not limited to, abrasion or fine needle aspiration.
  • the method comprises detecting a mutation in the patient or monitoring disease course of the patient's histiocytosis by the above methods; and selecting and/or applying a treatment or therapy based on the detecting or monitoring.
  • the method comprises
  • the patient had not previously undergone testing for the mutation, and a determination that a mutation is present is followed up by additional monitoring, either with or without treatment.
  • the patient prior to the testing, the patient was treated with a medicament that targets the product of the gene having the mutation.
  • kits for performing the above methods may include a specific binding agent that selectively binds to a BRAF mutation, and instructions for carrying out any of the method as described herein.
  • a two-step assay design was developed for a 28-30 basepair footprint in the target mutant gene sequence.
  • This assay design (and other assays known in the art) is useful for amplifying any size sequence in various tissues or bodily fluids, for example less than 400, less than 300, less than 200, less than 150 bp, less than 100 bp, less than 50 bp, less than 40 bp, less than 35 bp, or less than 30 bp.
  • FIG. 1 summarizes the assay design, which includes a first pre-amplification step to increase the number of copies of a target mutant gene sequence relative to wild-type gene sequences that are present in the sample.
  • the pre-amplification is conducted in the presence of a wild- type (non-mutant) suppressing "WT blocker” oligonucleotide that is complementary to the wild-type sequence (but not the mutant sequence) to decrease amplification of wild-type DNA.
  • the pre-amplification is performed with primers that include adapters (or "tags") at the 5' end to facilitate amplification in the second step.
  • the second step is additional amplification with primers complementary to the tags on the ends of the primers used in the first step and a TaqMan (reporter) probe oligonucleotide complementary to the mutant sequence for quantitative, digital droplet PCR (RainDance Technologies, Billerica, MA). Assay development
  • FIG. 2 shows PCR results for positive and negative controls.
  • Thresholds for mutation detection were determined by assessing data from 50 healthy controls and 39 patient samples using a classification tree. Minimizing the percentage of false negatives was given a higher importance than minimizing false positives.
  • a set of non-limiting thresholds for BRAF V600E were defined: ⁇ 0.05% as no detection or wild-type; the range of 0.05% to 0.107% as "borderline”, and >0.107% as detected mutation.
  • Results from the monitoring of the ratio of the BRAF V600E mutation relative to wildtype BRAF in urine samples are shown in FIG. 4, which indicates responsiveness to anakinra, followed by an increase in BRAF V600E with the cessation of therapy and then responsiveness to vemurafenib.
  • the assay indicates responsiveness to two therapeutic agents against ECD without limitation to the mechanism of action of each agent. Additionally, the assay indicates a loss of responsiveness when a therapy ended. This supports the use of the assay to indicate a lack of responsiveness, or lack of disease control, when no therapy, or an ineffective therapy, is used. Moreover, this demonstrates that the assay may be used to monitor a change in therapy as described herein.
  • the remainder of FIG. 6 shows a correlation between urinary cfDNA and plasma cfDNA in detecting BRAF wildtype, mutant, and unknown genotypes when using the methods disclosed herein. This shows that urinary cfDNA detection of histiocytosis mutations have a high correlation in performance between urinary cfDNA and both tissue samples and plasma from blood samples. Additional data from the above studies is provided in Janku et al., 2014.
  • cfDNA analysis facilitated identification of previously undescribed KRASG12S mutant ECD and dynamically tracked disease burden in patients treated with a variety of therapies. These results indicate that cfDNA BRAFV600E mutational analysis in plasma and urine provides a convenient and reliable method of detecting mutational status and can serve as a non-invasive biomarker to monitor response to therapy in LCH and ECD.
  • LCH Langerhans Cell Histiocytosis
  • ECD Erdheim-Chester Disease
  • circulating tumor cell-free DNA to both identify the BRAF V600E mutation and monitor response to therapy represents a potentially transformative development for these orphan diseases.
  • Examples 1-6 above demonstrate that BRAF V600E mutations could be detected in cfDNA (Janku et al., 2014), and the concordance of cfDNA BRAF mutational genotype with tissue mutational status in ECD and LCH.
  • Those Examples also demonstrate the ability of quantitative urine and plasma cfDNA analysis to detect dynamic changes in BRAF V600E mutation burden during treatment of disease.
  • Use of urine as a source of cfDNA offers significant advantages in sample stability and ease of serial collection.
  • Tissue biopsies were performed as part of routine clinical care, with the site of biopsy based on radiographic and/or clinical assessment of disease involvement. lOmL of blood and between 60-120mL of urine was collected at each time point. Plasma was separated from blood samples using standard techniques. All samples were de-identified, and operators performing plasma and urine cfDNA analyses were blinded to the tissue genotype and clinical characteristics of all patients.
  • Tissue mutational genotyping Initial BRAF tissue mutation testing was performed by a variety of methods as part of routine care in CLIA-certified molecular diagnostic laboratories at MSKCC, MDACC, or the institution from which the patient was initially referred. Tissue with a BRAF V600E mutation identified as part of these analyses was considered positive. For tissue to be considered negative for the BRAF V600E mutation for the purposes of this analysis, it was required to undergo further testing by a high sensitivity assay, either Sanger sequencing with locked nuclear acid (LNA) clamping or next-generation sequencing.
  • LNA locked nuclear acid
  • Plasma and Urine cfDNA extraction and analyses Plasma cfDNA was isolated using the QIAamp Circulating Nucleic Acid Kit (QIAGEN; Germantown, MD) according to the manufacturer's instructions. Urine cfDNA was isolated as previously described (Janco et al., 2014).
  • Urine and plasma cfDNA were quantified by a droplet digital PCR (ddPCR; QX-100, BioRad) assay to a 44bp amplicon of RNase P, a single-copy gene as previously described (Janco et al., 2014). Quantified DNA up to 60ng was used for mutation detection of BRAF V600E by droplet digital PCR and KRAS mutations at codons 12 and 13 of exon 2 by massively parallel sequencing.
  • ddPCR droplet digital PCR
  • a two-step PCR assay targeting a very short (31bp) amplicon was employed to enhance detection of rare mutant alleles in cfDNA.
  • the first step amplification was done with two primers flanking the BRAF V600E locus, where both primers contain non-complementary 5' tags that hybridize to second round primers.
  • a complementary blocking oligonucleotide suppressed wildtype BRAF amplification, achieving enrichment of the mutant BRAF V600E sequence in this step.
  • the second step entailed a duplex ddPCR reaction using FAM (BRAF V600E) and VIC (wildtype BRAF) TaqMan probes to enable differentiation of mutant versus wildtype quantification, respectively.
  • the RainDrop ddPCR platform (RainDance; Billerica, MA) was used for PCR droplet separation, fluorescent reading, and counting droplets containing mutant sequence, wildtype sequence, or unreacted probe.
  • the assay identified BRAF V600E mutation fragments detected as a percentage of detected wildtype BRAF.
  • the assay was simplified to a dichotomous classifier by combining both indeterminate and negative range as 'not detected' yielding a single cutoff of ⁇ 0.107 for not detected and >0.107 as detected.
  • This pre- specified single cutpoint of 0.107 was chosen given that positive and negative BRAF V600E status for ECD patients from a previous study was not within the indeterminate range (Janku et al., 2014).
  • wildtype BRAF patients with metastatic cancer were used to determine a threshold for detection of BRAF V600E mutations.
  • BRAF V600E values for this wildtype BRAF population were normally distributed and therefore a pre- specified cutpoint equivalent to three standard deviations (0.021%) above the mean of wildtype BRAF controls (0.031%) or >0.094% mutant to wildtype was considered positive for BRAF V600E12.
  • KRAS mutation detection For KRAS mutation detection (G12A/C/D/R/S/V, G13D), a two-step PCR assay similar to that described for BRAF V600E was employed with an initial 31 bp targeted region, except that during the second round, flanking primers were used to add patient specific barcodes and adaptor sequences necessary for massively parallel DNA sequencing per manufacturer's instructions (MiSeq, Mumina; San Diego, CA). Sequence reads were filtered for quality (Q-score>20) and verified as matching the target sequence (no more than 3 mismatches permitted outside the mutation region). For each sample, KRAS mutant sequences were tallied and the percent of mutant was computed.
  • the distribution of background signal in the wildtype population was observed not to conform to a normal distribution.
  • the median and median absolute deviation of a KRAS wildtype population was used to produce a "robust" z-score and a cutoff of greater than 4 z-scores above the median mutant signal count of the population (or > 0.0092%) was determined to be a positive result (Malo et al., 2006). This approach is approximately equal to the mean + 3SD threshold when the data is normally distributed (data not shown).
  • BRAF V600E mutational detection Concordance of tissue, plasma, and urinary assessment of BRAF V600E mutational detection was performed by calculating the kappa coefficient. Correlation of BRAF Y600E:BRAF wildtype ratios based on BRAF tissue genotype was performed using Mann-Whitney U test. A two-tailed p-value ⁇ 0.05 was considered statistically significant.
  • urinary cfDNA analysis identified 2 patients as being BRAF V600E mutant that were not known to have the BRAF mutation previously. Subsequent tissue biopsy was performed in these patients and identified the BRAF V600E mutation, allowing both patients to enroll in an ongoing phase II study of vemurafenib for BRAF V600E mutant ECD and LCH patients (NCT01524978). Thus, tissue-base genotyping resulted in 21/30 (70%) patients with definitive BRAF status compared to 30/30 (100%) using urinary cfDNA (FIG. 1A).
  • Urinary cfDNA analysis failed to detect the BRAF V600E mutation in 1/15 (6.7%) patient positive by tissue biopsy.
  • the urine and plasma utilized for cfDNA analysis were sampled while the patient was in active treatment with a BRAF inhibitor with ongoing reduction in disease burden, whereas the tissue genotyping was performed prior to treatment.
  • BRAF V600 Wildtype Patient 56.7% (17/30) of the patients enrolled in this study were identified as having a BRAF V600E mutation based on either tissue genotyping and/or cfDNA analysis. Identification of additional somatic mutations in BRAF V600E-wildtype patients is therefore of great importance for identifying targeted therapeutic strategies for those patients without BRAF mutations as well as for identifying markers to track disease in cfDNA.
  • One BRAF wildtype patient here was found to have a KRAS G12S mutation in tissue material taken from a cardiac ECD lesion (FIG. 11A-D). This mutation was also found to be present by cfDNA analysis in both plasma and urine (FIG. 1 IE and Table 5). Although NRAS mutations have been reported in ECD 18, KRAS mutations have never previously been reported in these disorders.
  • ECD ECD
  • urine as the source of cfDNA as reported here particularly facilitated routine serial monitoring of BRAF V600E allele burden. While somatic mutation detection has been performed in cfDNA of cancer patients previously, nearly all prior studies utilizing urinary cfDNA in cancer were restricted to patients with genitourinary malignancies (Casadiao et al., 2014a, b; Zhang et al., 2012). However, urinary cfDNA detection of BRAF V600E mutations mirrored closely the results from plasma cfDNA analysis here. Moreover, as shown in FIG. 10, urinary samples for cfDNA could be obtained on a weekly basis allowing for disease monitoring on an outpatient basis without the need for phlebotomy or other medical procedures. Previous studies indicate that DNA in urine can be stabilized for at least nine days (Zhang et al., 2012), whereas plasma requires processing within six hours for accurate assessment of cfDNA (Chan et al., 2005).
  • Combined use of tissue and cfDNA genotyping analyses also allowed the identification of a KRAS mutation in BRAF wildtype ECD patients (a mutation not previously described in ECD). Future interrogation of RAS mutations in tumor biopsies and cfDNA from BRAF wildtype histiocytic disorder patients may provide an additional somatic mutational biomarker and therapy options in this patient population.

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Abstract

La présente invention concerne des méthodes de détection d'une mutation chez un patient atteint d'un histiocytose. L'invention concerne également des méthodes de sélection et/ou d'application de traitement ou de thérapie pour un patient atteint d'un histiocytose. L'invention concerne en outre une méthode de traitement d'un patient atteint d'un histiocytose.
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