WO2010142467A1 - Procédé de surveillance d'une maladie provoquée par une translocation génomique - Google Patents

Procédé de surveillance d'une maladie provoquée par une translocation génomique Download PDF

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WO2010142467A1
WO2010142467A1 PCT/EP2010/003747 EP2010003747W WO2010142467A1 WO 2010142467 A1 WO2010142467 A1 WO 2010142467A1 EP 2010003747 W EP2010003747 W EP 2010003747W WO 2010142467 A1 WO2010142467 A1 WO 2010142467A1
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million
cells
translocation
patient
genomic
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Giovanni Porta
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Giovanni Porta
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to methods of monitoring, diagnosing and gaining insights into the status and prognosis of a disease generated by or derived from a genomic translocation in a cell or cells of a patient, in particular a chronic myeloid leukaemia patient, based for example on quantitative analysis of genomic DNA of leukaemic cells. It also extends to methods of treatment based on the results of the method.
  • Chronic myeloid/myelogenous leukaemia (CML also known as chronic granulocytic leukaemia (CGL)) is a clonal malignant myeloproliferative disorder which originates in a single haematopoietic stem cell. The progeny of this abnormal stem cell, immature granulocytes
  • the increased number of white blood cells in the bone marrow may result in low numbers of normal cells including red blood cells.
  • chronic myeloid leukaemia is characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow and the accumulation of these cells in the blood. It has been associated with chromosome anomaly called the Philadelphia chromosome. It is thought that 95% of patients with CML have this chromosomal abnormality.
  • Philadelphia chromosome originates from a reciprocal exchange of material between chromosome 22 and chromosome 9, i.e. by a translocation of genetic material.
  • the BCR gene breakpoint cluster region
  • the BCR gene breakpoint cluster region
  • part of the BCR ("breakpoint cluster region") gene from chromosome 22 (region ql l) is fused with the ABLl gene on chromosome 9 (region q34) to create a mutant fusion gene BCR-ABL located on chromosome 22.
  • Philadelphia chromosome can be described as t(9;22)(q34;ql l). Nevertheless, the exact break point of chromosome 9 and 22 inside the BCR and ABLl gene is unique to the individual.
  • the resulting BCR-ABL fusion gene encodes for a tyrosine kinase (fusion protein also known as the Bcr-abl fusion protein).
  • the molecular weight of the fusion protein expressed may be in the range 210 down to about 185 kDa. In CML this fusion protein usually is about
  • mutant fusion gene does not require activation by other cellular messaging proteins.
  • the mutant fusion gene activates a cascade of proteins which control the cell cycle, speeding up cell division.
  • protein produced by the mutant fusion gene inhibits DNA repair, causing genomic instability and making the cell more susceptible to developing further genetic abnormalities.
  • patients may be asymptomatic or have mild symptoms such as tiredness (fatigue), loss of appetite, weight loss, increased sweating, abnormal bruising and bleeding, a feeling of fullness or a tender lump on the left side of the abdomen, due to an enlarged spleen (splenomegaly). Swelling of the spleen may also cause pressure on the stomach, which can lead to digestive problems and poor appetite.
  • mild symptoms such as tiredness (fatigue), loss of appetite, weight loss, increased sweating, abnormal bruising and bleeding, a feeling of fullness or a tender lump on the left side of the abdomen, due to an enlarged spleen (splenomegaly). Swelling of the spleen may also cause pressure on the stomach, which can lead to digestive problems and poor appetite.
  • the accelerated phase there may be no more symptoms than in the chronic phase, however, the number of healthy blood cells in the blood may be lower and/or the number of leukaemic cells may be higher.
  • the WHO criteria are perhaps most widely used and define the accelerated phase by any of the following:
  • the accelerated phase is significant because it signals that the disease is progressing toward the blast crisis (blast phase). Although this may not cause any noticeable symptoms, some people may develop high temperatures (fever) and night sweats.
  • the blast phase may be characterised by:
  • Symptoms include:
  • Chronic myeloid leukaemia has an annual incidence of 1 to 1.5 per 100,000 of the population in Italy with about 800 new case being identified each year. In the United Kingdom about 700 new cases are identified each year. It is rare in children under 10 years of age as the onset is typically between 45 and 55 years of age.
  • Cytogenetics is relevant to the diagnosis, typically employing chromosomal analysis of bone marrow or blood samples via fluorescent in situ hybridisation (FISH).
  • FISH employs fluorescent probes to bind to specific targets in the BCR-ABL region of chromosome 9 and 22. The fluorescent probes bind only those parts of the chromosome with a high degree of sequence similarity thereto.
  • the labelled chromosomes are viewed under a microscope and the translocation identified. Usually multiple rounds of fluorescence in situ hybridisation are required to identify the position of the translocation. Thus FISH mapping is time-consuming and has limited resolution.
  • RT-PCR reverse transcriptase PCR
  • a number of PCR kits are available that measure the mRNA products of the BCR-ABL fusion gene. mRNA only contains transcribed exons from the genes, having already had the intronic material removed during RNA splicing. This means that information about the patient-specific break point has been removed.
  • the inventor believes that at certain stages of the disease leukaemic cells (i.e. cells containing a genomic translocation) may be present but are not in fact expressing mRNA product.
  • RT-PCR reverse transcriptase PCR
  • a method for monitoring a disease generated in a patient by a genomic translocation or providing a prognosis in relation to the disease comprising the step of: analysing an in vitro sample of at least 2,000 cells derived from said patient for the presence of a break point in genomic DNA in the sample cells, and/or the quantity of cells with a genomic break point, wherein the method employs direct one-step analysis of DNA from said sample cells.
  • Disease generated by a genomic translocation is intended to refer to a disease generated by the translocation, disease derived from the translocation or disease in some way closely associated with the existence and/or creation of the translocation.
  • diseases associated with a genomic translocation include: inflammatory myofibroblastic tumor; myelodysplastic syndrome; epithelioid hemangioendothelioma; myeloproliferative disorder, for example leukaemia such as ALL in particular T-cell acute lymphoblastic leukaemia, pre-B-cell acute lymphoblastic leukaemia, AML (acute myeloid leukaemia), chronic lymphocytic leukaemia CLL, multiple myeloma with eosinophilia, chronic myelomonocytic leukemia with eosinophilia; multiple myeloma; myxoid liposarcoma; lipoma; alveolar rhabdomyosarcoma; alveolar
  • MRD minimal residual disease
  • direct measurement and directly measure refer to the ability to identify the presence of a cell or cells containing a Philadelphia chromosome/fusion gene/genomic break point in a large number of cells and/or the number (quantity, percentage etc) of cells containing the translocation by a technique applied to DNA.
  • Direct measurement is NOT indirect measurement. The latter as employed herein refers to measuring the product of DNA, be it mRNA or protein.
  • a large number of cells as employed herein is intended to refer to at least 2,000 cells, for example 5,000 cells, 10,000 cells, 100,000 cells, 1 million, 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million, 10 million cells or more.
  • Monitoring refers to analysis of patients after they have been diagnosed with a relevant disease.
  • Prognosis as employed herein is intended to refer to predicting the patient's response or future development/progression of the disease, for example after treatment.
  • the method herein is NOT just the identification of the presence or absence of a breakpoint. Nevertheless the method according to the present invention can be adapted to provide a method of diagnosis with a high sensitivity and thus may be able to identify patients with an earlier stage of disease that are more suitable for treatment.
  • 000 cells, 10,000 cells, 100,000 cells, 1 million, 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million, 10 million, 15 million cells or more derived from said patient are analysed for the presence of a genomic break point therein.
  • One-step as employed herein is intended to refer to one discrete analysis without the requirement of human intervention, for example after addition of reagents the results would generally be available from the output of the instrument performing the analysis.
  • Multiple FISH analysis to increase the representative sample size is not intended to be a one-step analysis within the meaning of the present specification. Washing of CGH slides after the hybridization stage and/or prior to scanning is not intended to be a one-step analysis within the meaning of the present specification.
  • Microarray/CGH/chip technology provides an indication of whether ANY of the original cells in a sample had a genomic break point, not HOW MANY had the break point.
  • the hybridization process is a competitive process such that not all of the sample is represented in the results. Thus one cannot be certain that a negative result is not a false negative.
  • the technology could not be described as one-step in the context of the present specification. In one embodiment the invention does not encompass CGH technology other than its use as a means of identifying the location of the specific break point.
  • the method is qualitative in that it is able to analyse a large number of cells for the presence or absence of a genomic translocation, such as a leukaemic cell. In one embodiment the method is quantitative.
  • the method employs PCR on genomic DNA, in particular quantitative PCR.
  • DNA in PCR is less prone to degradation than RNA.
  • the method employs patient specific probes and/or primers, for example based on a patient's introns.
  • the method according to the present invention allows large numbers of cells to analysed for any one sample, for example 500, 1,000, 500,000, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 million cells or more can be analysed from a given sample. This makes the result of the analysis of the population of cells highly representative of the true number of cells containing a genomic translocation present in the patient. It is believed that if 10 million cells are analysed then one, for example leukaemic, cell in the population can be identified/quantified. Thus the method is also sensitive.
  • the method according the disclosure is able to identify 1 leukaemic cell in 10,000, for example 1 leukaemic cell in 100,000, 1 million, 1.5 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, 5.5 million, 6 million, 6.5 million, 7 million, 7.5 million, 8 million, 8.5 million, 9 million, 9.5 million or 10 million cells.
  • the method employs an accurate/precise method of quantifying cells containing a genomic translocation, for example leukaemic cells.
  • Accuracy as employed herein is intended to refer to the fact the number of false positive results and/or false negative results for a given sample is low, for example 1% or less.
  • Precision as employed herein is intended to refer the fact the methods according to the invention provide reproducible results, provided the conditions under which the experiments/analysis is performed are controlled (for example temperature etc). Clearly the type reagents and detectors employed will influence this parameter. In contrast employing cytogenetics, at most, less than 1,000 cells could be analysed from a sample. Furthermore, FISH is considered to have an error rate of 5-10%. Thus cytogenetics is not an accurate or sensitive method of quantifying the leukaemic cells or other cells containing a genomic translocation, in the context of the present specification.
  • FISH FISH cannot test a representative cell sample and RT-PCR suffers from the problems described above.
  • the inventor has performed a comparison of quantitative analysis using the two techniques (direct and indirect techniques) on patients with chronic myeloid leukaemia undergoing treatment with the approved medication imatinib and propose the following observations from the data: • the amount of gene products expressed from leukaemic cells varies (thus there is not a linear relation between the number of leukaemic cells and the amount of mRNA produced). For example, in Table 2 when 35% of cells were shown to be leukaemic using direct (DNA) measurement (patient 1), the amount of BCR- ABL/ ABL mRNA was 228%.
  • imatinib and other treatments such as dasatinib and nilotinib may provide long term remission for some patients, physicians have been reluctant to stop treatment because many patients simply relapse. Thus whilst it may be that as many as
  • a patient may be considered to be in remission if the result of the analysis is that: there are no cells identified with a genomic translocation, or the number of cells with a genomic translocation is constant or lower on one or more occasions (such as the result of 3 consecutive analysis over a 1 to 6 month period) than the results of a previous or initial analysis.
  • the method of the present invention may also be suitable for identifying those patients with the potential to respond well to therapy, for example those patients with a low percentage of cells with a translocation regardless of the amount of product expressed by those cells.
  • a method of identifying a chronic myeloid leukaemia patient in remission or not in remission comprising the step of quantitative assaying the amount of leukaemic cells in a sample derived from a patient, for example bone marrow or peripheral blood, by directly measuring the quantity of cells with a genomic break point which form a Philadelphia chromosome.
  • the method also provides a method of monitoring a patient with myeloid leukaemia and may provide valuable information on the likely prognosis and/or progression of the patient.
  • the assay is directed at the BCR-ABL gene in the Philadelphia chromosome.
  • the assay can employ any quantitative method for genomic DNA.
  • quantitative real time PCR Q-PCR is a particularly suitable technique because it is sensitive, robust and is widely available in laboratories throughout the world.
  • any of the above steps or alternative method for identifying the position of the break point may be employed in combination with the method of the present invention. Before the analysis to identify the break point is performed some sample preparation may be required, for example derivative chromosome isolation.
  • Chromosome sorting is performed for example on a fluorescence activated cell sorting (FACS) VantageTM (available from BD) cell sorter equipped with two lasers that emit in the UV range, for example at 330-360 nm and 457nm respectively, with 200 mW output each. Chromosome suspensions are stained with H33258 and
  • the purity of the sort is determined by extensive quality controls. Samples of sorted chromosomes are fixed on slides and hybridized with a probe specific for the sorted chromosome. Depending on the quality of the chromosome preparation a purity of more than
  • the DNA derived from the two chromosomes is then digested and used separately to hybridize a comparative genomics hybridization (CGH) slide.
  • CGH comparative genomics hybridization
  • the two hybridizations will indicate in each patient the sequences present on chromosome 22 flanking the sequence on chromosome 9.
  • the Philadelphia chromosome and its derivative are digested and hybridized to a slide such as a CGH slide, which enables the identification of the position of the break point.
  • a more efficient method is to employ microchips designed specifically to cover only the genomic region of interest. The advantage of this customized DNA chip apart from the price is that the spots will represent sequences laying 800 bp or less from each other.
  • the primers and/or the probes can be designed directly in the sequence across the break point from chromosome 9 to 22.
  • kits comprising a microarray chip designed to cover the chromosomes comprising a breakpoint, for example 9 and/or 22 translocation regions involved in forming a Philadelphia chromosome wherein the oligos laid down on the chip are up to 60 nucleotides long and the sequence coverage is no more than 160 base pairs apart and reagents and/or instructions for use in the analysis according to the present invention.
  • the length of the oligos is designed according to the target sequences. Generally the longer the oligos on the chip the more specific to the site they will be. Each oligo may be of the length 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28,
  • the primers and the probe are designed across the genomic break point of each patient.
  • the reason they are specific to each patient is because the chromosome breakage can occur in any region of the introns (non-coding regions) of, for example BCR and
  • the method according the disclosure herein includes the step of identifying where the translocation is situated for a given patient and optionally preparing primers to bind specifically in said location.
  • the primers and probe will remain relevant to a given patient, for as long as the disease is present and can then be used to monitor the number of cells (for example
  • Ieukaemic cells during the patient's therapy. This is particularly true when the technique used to monitor the cells is PCR.
  • the length of the primers/probes for PCR is designed according to the target sequences.
  • Each primer may be of the length 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
  • PCR can be performed in the usual way to identify and/or quantify the cells with the translocation.
  • the method may in addition contain the step of performing a PCR assay based on mRNA, for example RT-PCR.
  • a PCR assay based on mRNA for example RT-PCR.
  • kits include: BCR/ABL1 Quant (RUO) kit Asuragen Inc. a research tool utilizing multiplex real-time quantitative RT-PCR to provide simultaneous detection and quantification of BCR/ABLl fusion transcripts (b2a2, b3a2, and ela2), ABLl (an endogenous control), and BCR/ABLl Quant Norm (an exogenous control) in a single reaction.
  • BCR/ABL1 Quant (RUO) kit Asuragen Inc. a research tool utilizing multiplex real-time quantitative RT-PCR to provide simultaneous detection and quantification of BCR/ABLl fusion transcripts (b2a2, b3a2, and ela2), ABLl (an endogenous control), and BCR/ABLl Quant Norm (an exogenous control) in a single reaction.
  • the kit is based on TaqMan(R) technology and is compatible with ABI 7500 real-time systems or equivalent. Fusion transcripts corresponding to BCR/ABLl b2a2 or b3a2 are present in >95% of CML patients. About 5% of children with acute lymphoblastic leukaemia (ALL) and 20-35% of adult ALL also carry t(9;22), most often ela2.
  • This molecular assay provides sensitive quantitation of the BCR/ABLl transcript from RNA extracted from peripheral blood, bone marrow aspirates or cultured cells.
  • BCR/ABLl Quant is an assay providing broad target coverage and dynamic range with internal and external assay calibration powered by Asuragen's Armored RNA(R) Quant(TM) (ARQ) Technology.
  • the kit includes an optional exogenous internal spike in ARQ control, BCR/ABLl Quant Norm, to assess process efficiency and 4 external calibrators consisting of a blend of precisely quantified BCR/ABLl, ABLl and BCR/ABLl Quant Norm ARQs mixed at different concentrations to generate 3 standard curves.
  • the resulting PCR products are compatible with capillary electrophoresis (CE) for subsequent determination of the fusion transcripts identity (ela2, b2a2, or b3a2) via size fractionation.
  • CE capillary electrophoresis
  • the assay of the present disclosure can easily be performed on a sample derived from peripheral blood having the advantage of a less invasive sampling. Alternatively, a sample can be derived from a bone marrow biopsy from a patient.
  • Bone marrow samples can be taken daily, weekly, monthly, for example every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, as required. Bone marrow samples, realistically, can only be taken once a year because of the invasive nature of the testing.
  • the analysis of bone marrow samples may provide additional information in that it allows the analysis of the presence of abnormal stems cell, i.e. undifferentiated cells, which are primarily located in the bone marrow.
  • abnormal stems cell i.e. undifferentiated cells, which are primarily located in the bone marrow.
  • Zero abnormal stem cells provides a high level of confidence that the patient is in remission.
  • Analysis of a (one) patient derived sample may be performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, thereby allowing a larger number of cells to be analysed. Routinely, the analysis may be performed 2, 3 or 4 times on a sample.
  • the method optionally includes the step of analysing the results and assigning the patient to the category of in remission or not in remission, for example as defined herein above.
  • the method comprises the further step of stopping/suspending or reducing treatment for a disease associated with a genomic break point for a patient identified as remission or with mild residual disease.
  • the sensitivity of the genomic assay revealed the persistence of leukaemic cells at 0.001% (Table 2). Patients with levels of leukemic cells above this may be considered not to be in remission and therefore in need of further treatment. Patients in remission may generally have undetectable level of leukemic cells.
  • a method of treating a patient with chronic myeloid leukaemia wherein the patient is identified as in remission by a method herein comprising the step of stopping imatinib treatment (or corresponding treatment such as dasatinib or nilotinib) and optionally monitoring the patient for presence and/or the quantity of leukaemic cells.
  • the monitoring could be as frequent as the physician may decide because the samples analysed can be peripheral blood and thus it is not necessary to employ bone marrow samples. It may, for example be daily, weekly, monthly, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 monthly, such as 1 monthly initially but then may be moved to 3 monthly or 6 monthly. If the physician is confident that the remission has been sustained for a long period then the monitoring may be annually.
  • monitoring should be increased or maintained at short intervals because relapse can occur in these circumstances.
  • kits comprising the components for an assay according to the invention and/or instructions on how to perform the assay.
  • a method of diagnosing chronic myeloid leukaemia comprising the step of quantitatively assaying the amount of leukaemic cells in a patient from a sample, derived from bone marrow or peripheral blood, by directly measuring the leukaemic cells, analysing the results thereof and assigning the patient to the category of chronic myeloid leukaemia or non-chronic myeloid leukaemia.
  • a method of predicting the prognosis for a patient already identified as a chronic myeloid leukaemia sufferer comprising the step quantitative assaying the amount of leukemic cells in a patient from a sample, derived from bone marrow or peripheral blood, by directly measuring the cells with a break point which form a Philadelphia chromosome, analysing the results thereof and assigning a patient as likely to progress into remission following treatment, for example with imatinib, or as a patient likely to require long term maintenance treatment.
  • Table 2 for patient ID No: 1 may indicate that a low level of leukaemic cells, for example even in the presence of high levels of mRNA may render the patient particularly conducive to treatment, for example with imatinib, and therefore likely to go into remission.
  • the method of the present invention can also be used as a robust tool to monitor the effectiveness of new and/or experimental treatments for cancers such as chronic myeloid leukaemia, for example in clinical trials.
  • a lower dose for example 200-400mg/day of drug may be required for effective treatment or maintenance of the disease. If there are larger numbers of leukaemic cells then higher doses such as 600-800mg/day may be required.
  • the present method provides the opportunity to give the patient the most appropriate dose for their specific symptoms.
  • a method of treating a patient comprising the step of assessing accurately the number of cells with a translocation and then administering a dose of medicament, for example imatinib selected specifically for the patient based on the number of leukaemic cells identified.
  • the methods employed above for chronic myeloid leukaemia may also be employed to identify and monitor translocations in other tumours, cancers and diseases that result in hyper- functionality or gain of function of a new gene. Examples of other translations locations are:
  • Mucosa-associated lymphoid tissue (MALT) lymphoma is sometimes associated with a translocation t(l;2)(p22;pl2): related proteins are BCLlO and kappa light chain and/or translocation t(l;14)(p22;q32): related proteins BCLlO and IgH;;
  • Inflammatory myofibroblastic tumor is associated with translocation t(l;2)(q22- 25;p23): related proteins tropomyosin alpha-3 chain and ALK.
  • the translocation is rare in anaplastic large cell lymphoma;
  • Myelodysplastic syndrome is sometimes associated with translocation t(l;3)(p36;q21): related proteins are MELl and RPNl and/or translocation t(l;7)(ql ⁇ ;pl ⁇ ): proteins unknown; Epithelioid hemangioendothelioma is associated with translocation t(l;3)( P 36.3;q25);
  • T-cell Acute lymphoblastic leukaemia is associated with translocation t(l;7)(p34;q34): related proteins LCK and TCR beta;
  • Alveolar rhabdomyosarcoma can be associated with a translocation t(l;13)(p36;ql4): related proteins PAX7 and FKHR; and/or the translocation t(2;13); mantle cell lymphoma and pre-B-cell Acute lymphoblastic leukaemia may be associated with translocation t(l;14)(q21;q32): related protein BCL9 and IgH; pre-B-cell Acute lymphoblastic leukaemia may be associated with translocation t(l;14)(q25;q32): related proteins LHX4 and IgH and/or translocation t(l ;19)(q23;pl3.3): PBXl and E2A:- references: MoI Cell Biol 1994;14:3938; hyaline vascular Castleman's disease may be associated with translocation t(l;16)(pll;pll):- references: AJSP 2000:24:882 strange paro
  • Ewing's sarcoma/PNET may be associated with translocation t(l;22)(p36.1;ql2): ZSG and EWS genes:- references: Oncogene 2000:19:3799 follicular lymphoma may be associated with the translocation t(l;22)(q22;qll): FC gamma RIIb and lambda light chain:- references: OMIM 604590
  • Anaplastic large cell lymphoma may be associated with translocation t(2;3)(p23;q21): ALK and TFG (tropomyosin receptor kinase fused gene); follicular thyroid carcinoma and/or follicular adenoma may be associated with translocation t(2;3)(ql3;p25): related proteins
  • PAX8 and PPAR gamma 1 - references: AJSP 2002:26: 1016, Am J Path 2005:167:223; fibrous hamartomas may be associated with translocation t(2;3)(q31;q21): unknown proteins; reference Archives 2005:129:520; anaplastic large cell lymphoma (TYNK subtypes, 40-70%) and/or inflammatory myof ⁇ broblastic tumor may be associated with translocation t(2;5)(p23;q35): related proteins
  • ALK and NPM - references: Blood 1989;73:806 (early report), Blood
  • Burkitt's lymphoma and/or mantle cell lymphoma may be associated with translocation t(2;8)(pl2;q24): related proteins kappa light chain and c-myc:- reference Mod Path 2002; 15: 1266 (mantle cell lymphoma); REL and diffuse large cell lymphoma may be associated with translocation t(2,8)(pl2-16;q24):- references: Blood 2004:103:1862 sclerosing perineurioma may be associated with translocation t(2;10)(p23;q24): proteins unknown:- reference AJSP 2005:29:1164); fibroma of tendon sheathand/or and desmoplastic fibroblastoma/collagenous fibroma may be associated with translocation t(2;ll)(q31-32;ql2):- references Histopathology 1998:32:433, Cancer Genet Cvtogenet 2004:149: 161 ; alveolar rhabdomy
  • AJSP 2002:26:938; CLL and/or ALL and/or AML may be associated with translocation t(2;14)(pl3;q32): BCLl IA and IgH;- references: Leukemia 2002:16:937. Blood 2001:98:3413 (free).
  • Leuk Lymphoma 2002:43:2063 (case report); inflammatory myofibroblast ⁇ tumor and/or anaplastic large cell lymphoma may be associated with translocation t(2;17)(p23;q23): related proteins
  • ALK and CLTC - references: Am J Path 2001 :159:411 (free).
  • -Mod Path 2003:16:828: follicular lymphoma and/or CLL/SLL may be associated with translocation t(2;18)(pll-12;q21): related proteins kappa light chain and BCL2:- references: Leuk Lymphoma 1992:8:197.
  • CML and/or myelodysplastic syndrome may be associated with translocation t(3;21)(q26;q22): EVIl and AMLl :- references: Oncogene 2004:23:4263;
  • ALL and/or AML M4/M5 may be associated with translocation t(4;ll)(q21;q23): related protein AF4 and ALL1/MLL:- reference Ann Hematol 1992:65:143, Blood 2005:105:3434; multiple myeloma may be associated with translocation t(4;14)(pl6;q32): related protein FGFR3 and IgH:- references: Blood 2005; 105:4060. Clin Cancer Res 2004:10:5692;
  • ALL with eosinophilia may be associated with translocation t(5;9)(q31;p24): related proteins IL3 and JAK2 genes:- references Archives 2003;127:601 chronic myelomonocytic leukemia with eosinophilia may be associated with translocation t(5;12)(q33;pl3): PDGFRB and ETV6:- references: Acta Haematol
  • multiple myeloma and/or diffuse large B cell lymphoma may be associated with translocation t(6;14)(p21.1 ;q32.3); related proteins cyclin D3 and IgH:- references: Mod Path 2003:16:886 TGIST): multiple myeloma may be associated with translocation t(6;14)(p25;q32): related protein MUM/IRF4 and IgH:- references: Leukemia 1999; 13:1812
  • T-cell ALL may be associated with translocation t(7;9)(q34;q34.3): TCR beta and TAN1/NOTCH1:- references: Cell 1991 :66:649. Cancer Lett 2005:219:113; low grade fibromyxoid sarcoma, hyalinizing spindle cell tumor with giant rosettes t(7;16)(q34;pll): proteins unknown:- references AJSP 2003:27:1229. Archives 2000; 124: 1179; endometrial stromal sarcoma may be associated with translocation t(7;17)(pl5;q21): related proteins JAZFl and JJAZl:- references: AJSP 2004:28:224.
  • B-cell ALL may be associated with translocation t(8;9)(q24;pl3): related protein c-myc Archives 2003;127:610-ease report) myeloproliferative disorder s such as T cell lymphoblastic lymphoma may be associated with translocation t(8;13)(pll-12;qll-12): related proteins FGFRl and ZNF 198:- references: Nat Genet 1998:18:84.
  • Acta Haematol 2002:107:101; Burkitt's lymphoma and/or ALL-L3 and/or mantle cell lymphoma may be associated with translocation t(8;14)(q24;q32.3): related proteins c-myc and IgH:- references AJSP 2003:27:818 (Burkitt's in transplant recipients). Mod Path 2002:15:1266 (mantle cell lymphoma).
  • Leukemia 2003:17:585; AML-M2 with Auer rods and/or granulocytic sarcoma may be associated with translocation t(8;21)(q22;q22): related proteins ETO and AMLl :- references Nat Med 2002;8:743 (free), Proc Natl Acad Sci USA 2005;102:4016; Burkitt's lymphoma may be associated with translocation t(8;22)(q24;qll): elated proteins c-myc and lambda light chain;
  • AML-M5a and M4 and/or therapy related AML and/or ALL may be associated with translocation t(9;ll)(p22;q23): related proteins AF9 and MLL/ ALLl :- references Genes Chromosomes Cancer 1991:3:74, Hum MoI Genet 2000;9:1671 ; lymphoplasmacytic lymphoma and/or diffuse large B cell lymphoma and other B cell lymphoproliferative disorders may be associated with translocation t(9;14)(pl3;q32): related proteins PAX5 and IgH: reference Blood 1996:88:4110. Genes Chromosomes Cancer 2005:44:218.
  • Hum Path 2004:35:447 (not characteristic for lymphoplasmacytic lymphoma), Leuk Lymphoma 2000:36:435; extraskeletal myxoid chrondrosarcoma may be associated with translocation t(9;15)(q22;qll-q21): related proteins TEC/CHN and TCF12:- references: Am J Path 2003:162:781.
  • Cancer Res 2000:60:6832 myxoid chondrosarcoma may be associated with translocation t(9;17)(q22;qll- 12): related protein TEC/CHN and TAF2N/RBP56; [variant of t(9;22)]:- reference Cancer Res 1999:59:5064.
  • CML Genes Chromosomes Cancer 2002:35:340 CML (100%), preB ALL (5% of children, 25% of adults), and/or AML may be associated with translocation t(9;22)(q34;qll): related proteins c-abl and bcr (Philadelphia chromosome):- references: Mayo Clin Proc 2005:80:390. Clin Lab Sci 2005:18:38. more information (CML).
  • translocation t(10;14)(q24;qll) related proteins HOXI l and T cell receptor delta:- references: Proc Natl Acad Sci USA 1990:87:3161 (free).
  • Leuk Lymphoma 1995;16:209. low grade non-Hodgkin's lymphomas may be associated with translocation t(10;14)(q24;q32): related proteins NFKB-2/LYT10 and IgH;- references: CeU 1991;67:1075.
  • AML (frequent), ALL (10% involve 1 Iq23 rearrangements, less involve self- fusion) may be associated with translocation t(l 1 ;1 1)(q23;q23): MLL/ALL1 (self-fusion):- references: Proc Natl Acad Sci USA 1998:95:2390 (free). Leuk Res 2005:29:517, Blood 1996;87:2496 (free). Cancer Res 1997:57:117: T-cell ALL (5% of childhood cases) may be associated with translocation t(ll;14)(pl3;qll): related proteins rhombotin 2 (TTg-2, RBTN2) and T cell antigen receptor alpha/delta:- references: Leukemia 1995:9:1812;
  • T-cell ALL ( ⁇ 1%) may be associated with translocation t(ll;14)(pl5;qll): related protein rhombotin 1 (TTg-I /LMOl) and TCR alpha/delta; References: MoI Cell Biol 1989;9:2124; mantle cell lymphoma (90%), B cell prolymphocytic leukemia (20%, may represent mantle cell variant) and/or splenic lymphoma with villous lymphocytes (10%) and/or CLL (2-5%) and/or myeloma (2-5%) may be associated with translocation t(l I;14)(ql3;q32): BCLl/cyclin Dl and IgH:- reference Br J Haematol 2004; 125:330).
  • AML M3 variant may be associated with translocation t(ll;17)(q23;q21): related proteins PLZF and retinoic acid receptor-alpha:- references: Semin Hematol 2001 :38:37. Proc Natl Acad Sci USA 1997;94: 10255; MALT lymphoma (50%); also diffuse large B cell lymphoma may be associated with translocation t(ll;18)(q21;q21): related proteins API2 and MALTl:- references: Mod Path 2003; 16: 1232 (colorectal lymphomas), Int J Hematol 2005:82:59 (cytologic specimens);
  • AML and/or M4/M5; also M1/M2 may be associated with translocation t(ll;19)(q23;pl3): related protein ALLl and ELL:- references: Proc Natl Acad Sci USA 1994:91 : 12110, Cancer Genet Cvtogenet 2001:129:17 (case report); desmoplastic small round cell tumor may be associated with translocation t(ll;22)(pl3;ql2): related proteins WTl and EWS:- references: AJSP 2002:26:823. Archives 2002:126:1226 (lung tumor) [correction at Archives 2003:127:7821. Mod Path 2002:15:673 (dural tumor). AJSP 1992; 16:411 (original report). Semin Cancer Biol 2005;!
  • Ewing sarcoma/PNET (90% of cases) may be associated with translocation t(ll;22)(q24;ql2): related proteins FLIl and EWS: references: Adv Anat Pathol 2005:12:212. Cancer Res 2005;65:4633;
  • translocation in i(12p) may be associated with intratubular germ cell neoplasia; germ cell tumors:- references: Mod Path 2005:18 Suppl 2:S51.
  • APMIS APMIS
  • pulmonary chondroid hamartomas may be associated with translocation t(12;14)(ql4-15;q23-24): related proteins HMGA2/HMGIC and various:- references: Cancer Genet Cvtoeenet 1988:32:13 (uterine leiomyomas), Cancer Genet Cytogenet 2002; 138:50 (various smooth muscle tumors).
  • Mod Path 2002:15:351 Intravenous leiomyomatosis), more information-HMG A2 ; AML and/or infantile (congenital) fibrosarcoma, cellular mesoblastic nephroma, secretory carcinoma of breast may be associated with translocation t(12;15)(pl3;q25): related proteins ETV6 and NTRK3; (Genes Chromosomes Cancer 2004:40:152), (Blood 1999:93:1355 (case report). Mod Path 2000:13:29, Mod Path 2001:14:1246, AJSP 2000:24:937.
  • more information-CHOP/DDIT3: preB-cell ALL (20%) may be associated with translocation t(12;21)(pl2- 13;q22): related protein TEL/ETV6 and AML1/CBFA2:- references: Curr Opin Hematol 2002:9:345, Diagn MoI Path 2000:9:184; AML may be associated with translocation t(12;22)(pl3;qll-12): related proteins TEL/ETV6 and MNl :- references: MoI Cell Biol 2000:20:9281. more information, MNl -more information.
  • myxoid liposarcoma may be associated with translocation t(12;22)(ql3;ql2): related proteins CHOP and EWS:- references: J MoI Diagn 2002:4:164, Clin Cancer Res 2000:6:2788 ), more information-CHOP/DDIT3; clear cell sarcoma of soft parts (>95%) may be associates with translocation t(12;22)(ql3;ql2): related proteins ATFl and EWS;
  • Mesenchymal chondrosarcoma may be associated with translocation der(13;21)(ql ⁇ ;ql ⁇ ): proteins unknown: references Mod Path 2002;15:572
  • Diffuse large cell lymphomas (4%) may be associated with translocation t(14;15)(q32;qll-13): related proteins IgH and BCL8:- references: Proc Natl Acad Sci USA 1997;94:5728; multiple myeloma (10%) may be associated with translocation t(14;16)(q32;q23): related proteins IgH and c-maf:- references: Blood
  • CLL/SLL (5%) may be associated with translocation t(14;19)(q32;ql3): related protein IgH and BCL3:- references: Leuk Lymphoma 2002:43:813, Genes Chromosomes Cancer 1997:20:64
  • AML-M3 acute promyelocy e leukemia
  • Genes Chromosomes Cancer 1999:26:265: multiple myeloma may be associated with translocation t(16;22);(q23;qll): related proteins c-maf and Ig lambda:- references: Blood 1998:91 :4457
  • CLL/SLL may be associated with translocation t(18;22)(q21;q21): lambda light chain (22ql l) and BCL2 (18q21):- references: Genes Chromosomes Cancer 1993;6:39, Genes Chromosomes Cancer 1991;3:205, Leukemia 1996;10:970
  • myelodysplasia syndrome myeloproliferative disorders and/or AML
  • myelomas may be associated with a translocation in region 2Oq:- reference Genes Chromosomes Cancer 2004;41 :223, Cancer Genet Cytogenet 2005:160:188; trisomy associated with desmoid-type fibromatosis and/or patients at risk for lung cancer may have a link with a translocation is region 20: Am J Path 1999:154:729. Int J Cancer 1995:63:527, Cancer Epidemiol Biomarkers Prev 1998:7:1051
  • Ewing's sarcoma/PNET (5-10%) and/or desmoplastic small round cell tumor may be associated with translocation t(21;22)(q22;ql2): related protein ERG and EWS:- AJSP 1998:22: 1026, Nat Genet 1994:6: 146, J Clin Oncol 1999:17:1809
  • Translocations - chromosome 22 CML variant Philadelphia chromosome may be associated with translocation t(22;22)(ql3;ql 1): related protein bcr:- references: Leuk Res 1986:10:1131, Blut
  • Certain subtypes of renal cell carcinoma may be associated with translocation t(X;l)(pll.2;q21.2): related protein TFE3 and PRCC:- references: AJSP
  • anaplastic large cell lymphoma may be associated with translocation t(X;2)(qll;p23): related proteins MSN and ALK; subungual exostosis; also cases reports of AML-M7, premature ovarian failure, female with Duchenne muscular dystrophy may be associated with a translocation in region t(X;6):- references: AJSP 2004:28: 1033 pediatric renal carcinoma, alveolar soft parts sarcoma may be associated with translocation t(X;17)(pll.2;q25): related protein TFE3 and ASPL:- references:
  • the disclosure provides a method of diagnosing or monitoring a tumor, cancer or disease resulting from a hyperfunction generated by a cell with a translocation, comprising the step of directly identifying and/or quantifying the cell with translocation, wherein the method does not employ cytogenetics to identify a translocation which forms the
  • Chromosome extraction from periferal blood and bone marrow in order to define chromosomal break points. 12 ml of peripheral blood (PBS) or 4 ml of bone marrow (BM) are collected in eparine or in anticoaugulants other then EDTA that inhibits cells growth.
  • PBS peripheral blood
  • BM bone marrow
  • 0.5, 1, 2, 3 ml of PBS or BM are cultivated in 8 ml of media (e.g. RPMI) for 24 hrs, 48 hrs and 72 hrs at 37 0 C and 5 % CO 2 .
  • media e.g. RPMI
  • colchicines e.g. l ⁇ l per ml of culture.
  • the cell suspensions are pooled together into one tube after resuspending the cell pellets in hypotonic solution.
  • the suspension of swollen cells is centrifuged.
  • the cell pellet is resuspended in ice-cold polyamine isolation buffer.
  • Chromosome suspention is applied to the flow cytometer.
  • Genomic junctions of BCR-ABL had been previously characterised in Mattarucchi et al. (Mattarucchi). Briefly, DNA from patients designated as 1, 2, 3, 4, 6, 8, 9 and 10 was extracted from blood or bone marrow, fragmented, ligated to adaptors, and amplified by a nested PCR using a BCR specific forward primer. Thus, genomic breakpoints were sequenced. The majority of patients were treated with IM monotherapy at a starting dose of 400 mg/day, except for patient #9 who was participating in a 800 mg/day trial. The dose for patients #6 and #10 was increased to 600 mg/day, as a consequence of suboptimal cytogenetic findings observed at 12 and 6 months, respectively.
  • Cytogenetic and molecular monitoring A cytogenetic analysis of bone marrow (approximately 20 metaphases for each patient) was performed before treatment using conventional QFQ-staining. Cytogenetic tests were repeated every 6 months, until a complete response was achieved. Then bone marrow metaphases were analyzed less frequently. Levels of BCR-ABL mRNA were measured upon diagnosis and approximately every 3 months thereafter. Molecular investigations were carried out at the reference laboratory of the CML network of the Italian Group of Hematological Malignancies in Adults (GIMEMA) at the Hospital of Bergamo, Italy. All analytical procedures were subject to the quality control process according to the ISO 9001 :2000 accreditation of the laboratory.
  • Each assay comprised two real time reactions: one directed against the breakpoint sequence (present in 1 copy, only in leukemic cells) and a second against the BCR sequence used as control (1 copy in leukemic cells and 2 copies in normal cells).
  • LC percentage of leukemic cells
  • the reaction mixture contained: 12.5 ⁇ l of the TaqMan® Universal PCR MasterMix (Appliedbiosystems, Foster City, CA, USA); 900 nM of each primer; 200 nM of probe; DNA ranging from 100 ng to 300 ng depending on the sample availability; and nuclease free water up to 25 ⁇ l.
  • the PCR thermal profile was: 2 minutes at 50 °C followed by 10 minutes at 95 °C and 45 amplification cycles (95 °C for 15 seconds and 60 °C for 60 seconds). Reactions were prepared and run in triplicate on ABI Prism 7000 SDS (Appliedbiosystems) and each experiment was repeated and confirmed a second time.
  • primers are designated as follows: F, common forward primer; Ph-R, reverse primer for the selective amplification of the BCR-ABL sequence; wt-R, reverse primer for the selective amplification of BCR.
  • results are reported as follow: top, percentage of BCR-ABL/ABL mRNA; bottom, percentage of leukemic cells. Results at the onset are approximated to the unit. --, unavailable data; UND, undetectable levels; *, bone marrow samples; f, peripheral blood samples.

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Abstract

La présente invention porte sur des procédés de surveillance, de diagnostic et d'obtention d'aperçus du statut et du pronostic, en particulier, de patients atteints d'une leucémie myéloïde chronique sur la base, par exemple, d'une analyse quantitative de l'ADN génomique de cellules leucémiques. Elle s'étend également à des procédés de traitement sur la base des résultats du procédé.
PCT/EP2010/003747 2009-06-09 2010-06-08 Procédé de surveillance d'une maladie provoquée par une translocation génomique WO2010142467A1 (fr)

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EP2536854A2 (fr) * 2010-02-18 2012-12-26 The Johns Hopkins University Biomarqueurs tumoraux personnalisés

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WO2009062166A2 (fr) 2007-11-08 2009-05-14 University Of Washington Identification et mappage sur la base d'une puce à adn de points de cassure de translocation équilibrée

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EP2536854A2 (fr) * 2010-02-18 2012-12-26 The Johns Hopkins University Biomarqueurs tumoraux personnalisés
EP2536854A4 (fr) * 2010-02-18 2013-08-07 Univ Johns Hopkins Biomarqueurs tumoraux personnalisés
US9957572B2 (en) 2010-02-18 2018-05-01 The Johns Hopkins University Personalized tumor biomarkers
US10900088B2 (en) 2010-02-18 2021-01-26 The Johns Hopkins University Personalized tumor biomarkers

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