CA2842635A1 - Pre-cancerous cells and their identification in the prevention and treatment of cancer - Google Patents

Abstract

There is provided a method for detecting evidence of pre-cancerous cells in a population of cells from a patient sample, the method comprising: screening the population of cells for a DNMT3a or IDH2 mutation; determining positive evidence of pre-cancerous cells if the population comprises cells containing a DNMT3a or mutation.

Description

PRE-CANCEROUS CELLS AND THEIR IDENTIFICATION IN
THE .PREVENTION AND TREATMENT OF CANCER
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No, 61/929,420 filed on January 20, 2014.
FIELD OF THE INVENTION
The invention relates to pre-cancerous cells and more particularly to identifying pre-cancerous cells in the prevention and treatment of cancer.
BACKGROUND OF THE INVENTION
There is overwhelming evidence that virtually all cancers are clonal and represent the progeny of a single ceIl. However, the evolutionary trajectory that leads from the first somatic mutation to the eventual development of cancer is not well mapped. The simplest models predict that each newly acquired somatic mutation confers selective advantage to drive successive waves of clonal expansion, with the fittest clone becoming dominant However the modern era of cancer genomics has exposed a more complex clonal architecture in many tumor typesg, where multiple genetically distinct out:clones co-exist with the dominant clone. Comparison of diagnostic and recurrent/metastatic samples obtained from the same patient has established that the latter frequently do not evolve from the dominant clone, but instead can be traced either to a minor subclone present at diagnosis, or to a putative, undetected ancestral clone'. Thus, a clear understanding of the genomic landscape of tumors is required in order to devise targeting strategies that eliminate not only the dominant clone but also the subcional reservoirs from which recurrence can arise.
Although the clonal composition of cancer lineages within individual tumors is coming into focus, the very first steps in cancer development remain poorly defined.
Early and possibly initiating mutations have been identified from analysis of pre-neoplastic DOCSTOR: 2935116\3 lesions in breast/1, lung, ski 'n111, and colon cancer, as well as from studies of AML
cases that evolved from a prior myelodysplastic syndrome (MDS). However, key questions remain unanswered. In particular, can clinically relevant clones be traced back to a non-tumorigenic cell? Do pre-cancerous ancestral clones persist after tumor development? If so, are they present in the diagnostic sample, and do they survive treatment and persist in remission samples?
Human leukemia is a disease model particularly suited to addressing these fundamental questions, due to the depth of our understanding of normal hematopoiesis and the availability of functional assays and analytic tools that allow examination of phenotypically defined populations at the single cell level. In AML, a subset of cases evolve from a preceding clinically overt phase such as MDS or chronic myeloid leukemia (CML), characterized by clonal expansion of one or more blood lineages'. The founder mutations present in pre-leukemic cells are retained in the AML blasts, implicating them as putative initiating events and establishing clonal 16 expansion as the first step in leukemogenesis. Interestingly, somatic mutations in some leukemia-associated genes such as TET2 have also been linked to multilineage clonal hematopoiesis in aging healthy individuals, Insight into the phenotype of the normal cell from which clonal expansion can initiate was first provided by the pioneering studies of Fialkow in CML, which demonstrated that BCR-ABL1 arises in a multipotential HSC2¨. However, for the majority of AML cases that arise de novo without any prior clinical perturbations, insight into the cellular context and functional consequences of the earliest genetic lesions requires identification and examination of ancestral cells within the diagnostic sample. Recent studies have found that only a subset of mutations contained in AML blasts were present in HSC-enriched cell 26 fractions isolated from AML patient samples, and that these cells were capable of non -leukemic differentiation26.27.
SUMMARY OF THE INVENTION
In an aspect, there is provided a method for detecting evidence of pre-cancerous cells in a population of cells from a patient sample, the method comprising:
screening the population of cells for a DNMT3a or IDH2 mutation; determining positive evidence of pre-cancerous cells if the population comprises cells containing a DNMT3a or 113[12 mutation.
DOCSTOR: 29351 16 \.3 2 In an aspect, there is provided a method of cancer therapy or cancer prevention in a patient, the method comprising providing a treatment to the patient that at least partially eliminates cells containing at least one of a DNMT3a or IDH2 mutation. In other aspects, the treatment could also reduce/eliminate the cancerous properties of such cells non-cancerous, or reduce/eliminate their potential to become cancerous.
In an aspect, there is provided a method of assessing a patient for risk of cancer, the method comprising: screening cells from a patient sample for mutations in DNMT3a or IDH2; and determining a patient is at risk of cancer if the sample contains cells containing the DNMT3a or IDH2 mutation.
In an aspect, there is provided a method of monitoring cancer therapy in a patient, the method comprising: screening cells from a patient sample for mutations in DNMT3a or 1DH2; and determining an effectiveness or progression of therapy, wherein effectiveness or progression is directly related to a decrease in cells in the sample containing the DNMT3a or I0H2 mutation.
In an aspect, there is provided a composition for use in cancer therapy or cancer prevention, the composition comprising a compound that at least partially eliminates cells containing at least one of a DNMT3a or IDH2 mutation, along with a pharmaceutically acceptable carrier.
In certain examples, Applicants establish that ancestral preL-HSC present at diagnosis are able to regenerate the entire hematopoietic hierarchy while possessing competitive repopulation advantage over non-leukemic HSC leading to clonal expansion.
These preL-HSC are found in a high proportion of AML patients that carry mutations in DNMT3a and IDH2, and unlike AML blasts, they survive induction chemotherapy and persist in the bone marrow at remission, providing a potential reservoir for leukemic progression.
BRIEF DESCRIPTION OF FIGURES
These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:
DOCSTOR: 293511613 Figure 1 shows recurrent somatic DNMT3a mutations are common in T-cells from AML patients. a, Summary of the allele frequency (%) of missense and frameshift somatic single nucleotide variants (sSNV) in AML-related genes assessed by deep targeted sequencing (read depth 250x) in AML blasts and T cells from the peripheral blood of 12 AML patients. The sSNV numbers indicated at the top of the table correspond to the numbers in Table 2. Somatic mutations in DNMT3a R882H; t, R137C) were found in both T-cells and AML blasts in Patients #9, 11 and 12.
Patient #12 also had a low frequency 1DH2 mutation (T, R140L) in T cells, b, Frequency (%) of DNMT3a"" and NPMlc in freshly isolated GD33+ blasts (AML) and matched 1-cell controls from 17 patients with normal karyotype AML, as determined by droplet digital PCR. For a and b, the length of the bars is proportional to the mutant allele frequency (the scale bar under the first column applies to all columns).
Figure 2 shows DNMT3a mutation precedes NPM1 mutation in human AML and is present in stem/progenitor cells at diagnosis and remission. a, Flow cytometric analysis showing the gating strategy used to isolate phenotypically normal stem and progenitor cell populations from AML patient samples. Plots show analysis of samples from Patient #11: diagnosis (day 0, peripheral blood mononuclear cells), remission (day 62, CD34+ enriched bone marrow) and relapse (day 379, peripheral blood mononuclear cells). b, Allele frequency of DNMT3a and NPMI mutations in stem/progenitor, mature lymphoid and blast (CD45dim C033+) cell populations, as indicated, isolated from diagnosis (gray), remission (white) and relapse (black) samples of Patient #11 as determined by droplet digital PCR (ddPCR). At remission, CD33+ myeloid cells were also analyzed. c, Summary of the occurrence of DNMT3an"
and NPM10 in isolated stem/progenitor, mature and blast cell populations from patient peripheral blood samples as determined by ddPCR, White, DNMT3a'' or NPM1c not detected; gray, DNMT3a mut alone; black, DNMT3a"1 + NPM1c. NA, no population detected; HSC, hematopoietic stem cell; MPP, multipotent progenitor; MLP, multilymphoid progenitor; GMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MEP, megakaryocyte erythroid progenitor; NK, natural killer cells. d. Graphic representation of DNMT3a' rm allele frequency in sorted cell populations isolated from diagnosis (0 months), early (3) and late (36) remission samples of Patient #57.
Figure 3 shows Pre-leukemic HSC bearing DNMT3ed generate multilineage engraftment and have a competitive advantage in xenograft repopulation assays.
a, DOCSTOR! 2935116 \ 3 4 Representative flow cytometric analysis of engrafted human cells harvested from NSG
mouse bone marrow (BM) 16 weeks after intrafemoral (i.f.) transplantation of peripheral blood mononuclear cells (PB MNC) from diagnosis and relapse samples of Patient #11, b, Analysis of human graft composition in NSG mouse BM 16 weeks after 6 If. transplantation of PB MNC from the diagnosis sample of Patient #11 across a range of cells doses. The percentage of human (CD46+) B (CD19+) and myeloid (CD33+) cells was determined by flow cytometry. Mutant allele frequency (1)/0) in the human graft was determined by droplet digital PCR (ddPCR) analysis of sorted human cells. The length of the bars is proportional to the mutant allele frequency (the scale bar under the first column applies to all columns). c, Summary of DNMT3dnut allele frequency in the human graft from mice analyzed by ddPCR 8 and 16 weeks after transplantation of PB MNC from Patient #11, compared to isolated hematopoietic stem celI/multipotent progenitors (HSC/MPP) from the patient's PB at diagnosis. *, P<0.05.
Bars indicate mean and standard deviation.
Figure 4 shows the identification of preL-HSC with IDH2 mutation. a, Summary of the occurrence of mutations in NPM1, DNMT3a, and IDH1/2 determined by Sanger sequencing, in AML patient peripheral blood samples (n=25) that generated a non-leukemic multilineage graft after transplantation into immune-deficient mice.
b, Representation of the proportion (%) of AML patient samples with DNMT3a and/or IHD1/2 mutations among samples that generated a non-leukemic multilineage graft in xenotransplanted mice. c, IDH2 and NP/V/1 mutant allele frequency (%) in stem/progenitor, mature lymphoid and blast (CD45dimCD33+) cell populations isolated from the peripheral blood of Patients #52, 64 and 77 at diagnosis, as determined by droplet digital PCR (ddPCR), Blank boxes indicate no mutation detected.
Figure 5 shows FLT3-1TD is a late event in patients carrying DNMT3a mutation.
PCR
analysis of FLT3-1-1012 in stem/progenitor, mature lymphoid and blast (CD45dim CD33 ) cell populations from Patient #13 (a) and #14 (b). FLT3-ITD was present in the blasts from both patients, and also in MLP from Patient #14. In contrast, DNMT3am11t without FLT3-1TD was detected in multiple non-blast cell populations (see also Figure 6). HSC, hematopoietic stem cell; MPP, multipotent progenitor;
CMP, common myeloid progenitor; MLP, multilymphoid progenitor; GMP, granulocyte monocyte progenitor; NK, natural killer cells.
DOCSTOR: 2935116 13 Figure 6 shows frequent occurrence of DNMT3a mutation without NPM1 mutation in stem/progenitor and mature lymphoid cells in AML patients at diagnosis. a, Summary of the allele frequency (%) of DNMT3a and NPM1 mutations in stem/progenitor, mature lymphoid, and blast (CD45dim CD33+) cell populations from 11 AML
patient peripheral blood samples obtained at diagnosis, as determined by droplet digital PCR
(ddPCR). Phenotypically normal cell populations were isolated by fluorescence activated cell sorting according to the strategy depicted in Fig. 2a. Mutant allele frequency ¨50% is consistent with a heterozygous cell population. Departures from 60% mutant allele frequency may be stochastic, related to clonal heterogeneity, or due to the presence of copy number variations, for example loss of the wild type allele (loss of heterozygosity) or amplification of the mutant allele. NA, no population detected; HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte erythroid progenitor; MLP, multilymphoid progenitor; GMP, granulocyte monocyte progenitor; NK, natural killer cells.
Blank boxes indicate no DNMT3a or NPM1 mutation detected. b, Representative plots showing ddPCR analysis of DNMT3amut and NPM1c allele frequency in sorted cell populations from Patient #11, The mutant allele frequency (%) is indicated on each plot.
Figure 7 shows phenotypically normal stem/progenitor and mature cell populations are present in AML patient samples at diagnosis, remission and relapse. Flow cytometric analysis showing the gating strategy used to isolate phenotypically normal stem/progenitor and mature lymphoid cell populations from AML patient samples.

Diagnosis and relapse samples are from peripheral blood; remission samples are from bone marrow.
Figure 8 shows cells bearing mutations in DNMT3a but not NPM1 are present at diagnosis in AML patients and persist at remission and relapse. Allele frequency of DNMT3a and NPM1 mutations of patients #28, 35, 55, and 57 in stem/progenitor, mature and blast (CD45dim CID33+) cell populations, as determined by droplet digital PCR (ddPCR). Cells were isolated from diagnosis (light gray), early remission (white), relapse (dark gray with prominent black outer circle) or late remission (light gray with prominent black outer circle) samples. At remission, CD33+ myeloid cells were also analyzed. I-1SC, hematopoietic stem cell; MPP, multipotent progenitor; MLP, multilymphoid progenitor; CMP, common myeloid progenitor, GMP, granulocyte DOC STOR: 2935116\3 , monacyte progenitor; MEP, megakaryocyte erythroid progenitor; Nit, natural killer cells.
Figure 9 shows PreL-HSC generate multilineage human grafts in immune-deficient mice in the peripheral blood of AML patients. Summary of results of limiting dilution experiments to assess frequency of preL-HSC generating multilineage grafts after xenotransplantation. Cohorts of NSG mice were transplanted intrafemorally with varying numbers of peripheral blood mononuclear cells from diagnostic samples of AML Patient #11 (a) and #55 (b) and analyzed after 8 or 16 weeks by flow cytometry.
Engraftment was defined as >0.1% human CD45+ cells in the injected right femur.
Shown is the number of mice with multilineage human grafts containing both CD33+
myeloid cells and CD33¨CD19+ cells. The frequency of preL-HSC was calculated using the ELDA platform.
Figure 10 shows frequent generation of non-leukemic multilineage human grafts in following xenotransplantation of peripheral blood cells from AML patients.
Summary of xenograft characteristics in 123 sublethally irradiated NSG mice transplanted intrafemorally with mononuclear peripheral blood cells from 20 AML patients at diagnosis and analyzed after 8 weeks by flow cytometry. The proportion of myeloid (CD33+) and B-lymphoid (CD33¨CD19+) cells in the human (C045+) graft is shown.

Leukemic (AML) engraftment is characterized by a dominant myeloid (CD45dimCD33+) graft, whereas non-leukemic multilineage grafts contain both lymphoid (predominantly CD33¨CD19+ B cells) and myeloid (CD33+) cells. No leukemic or multilineage graft could be detected in 65/123 mice (53%) in this cohort horizontal box indicates AML grafts (27 mice, 22%); vertical box indicates multilineage grafts (31 mice, 25%).
BRIEF DESCRIPTION OF TABLES
Table 1 shows the clinical characteristics of AML patients analyzed by targeted sequencing for leukemia-associated genes. nd, not done: MDS, myelodysplastic syndrome; FAB, French-American-British; WBCC, white blood cell count; BM, bone marrow; ABMT, allogeneic bone marrow transplant.
DOCSTOR: 2935116\3 Table 2 shows somatic single nucleotide variants (sSNV) of AML samples analyzed by targeted sequencing. Condel software was used to predict the effect of sSNV on amino acid changes, POS, position; ID, dbSNP ID from NCB1; REF, sequence from reference human genome build hg19; ALT, alternative sequence.
Table 3 shows clinical characteristics, xenograft characteristics and results of Sanger sequencing for 71 AML patients. Xenograft characteristics describe the human graft (human CD45+ >0.1%) generated after intrafemoral transplantation of 5x10 peripheral blood cells from AML patients into cohorts of sublethally irradiated NOD-S= (with anti-CD122 antibody treatment) or NSG mice. In this xenograft model, leukemic (AML) engraftment is characterized by a dominant myeloid (CD45dimCD33+) graft, whereas non-leukemic grafts are multilineage and contain both lymphoid (predominantly CD19+ B cells) and myeloid (CD33+) cells. nd, not done; MDS, myelodysplastic syndrome; FAB, French-American,British; WBCC, white blood cell count; BM, bone marrow; CR, complete remission; ABMT, allogeneic bone marrow 18 transplant; T, grafts containing >10% CD3+ cells, DETAILED DESCRIPTION
In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.
In non-limiting examples, Applicants investigate acute myeloid leukemia (AML) to determine the cell and mutations of origin that give rise to cancer. In AML, the cell of origin, nature and biological consequences Of initiating lesions and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukemic phase. Here, highly purified hematopoietic stern cells (HSC), progenitor and mature cell fractions from the blood of AML
patients were found to contain recurrent DNMT2a mutations (DNMT3amm) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts, DNMT3amuf-bearing HSC exhibited multilineage repopulation advantage over non-mutated HSC
in xenografts, establishing their identity as pre-leukemic-HSC (preL-HSC). preL-HSC
were found in remission samples indicating that they survive chemotherapy, Thus DNMT3amut arises early in AML evolution, likely in HSC, leading to a clonally expanded DOCSTOR: 2935116\3 pool of preL.-HSC from which AML evolves, Applicants' findings provide a paradigm for the detection and treatment of pre-leukemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.
In an aspect, there is provided a method for detecting evidence of pre-cancerous cells in a population of cells from a patient sample, the method comprising:
screening the population of cells for a DNMT3a or IDH2 mutation; determining positive evidence of pre-cancerous cells if the population comprises cells containing a DNMT3a or mutation.
A person skilled in the art would understand that although certain specific examples are directed to the direct detection of pre-leukemic stem cells, detection of a DNMT3a or IDH2 mutation in any cell downstream in the hierarchy could provide evidence of the mutation in the originating stem cell.
The term "sample" as used herein refers to any fluid, cell or tissue sample from a subject that can be assayed for the purposes described herein, for example, to determine the presence of a DNMT3a or IDH2 mutation in cells of the patient.
Preferably the sample is a blood sample.
In some embodiments, the patient sample is from a patient that has not been diagnosed with cancer, optionally, the patient is subclinical for cancer, Clinical diagnosis of cancer, such as leukemia, are well known to a person skilled in the art.
As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth, for example, resulting in tumours. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia, More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
DOCSTOR: 293511613 In preferred embodiments, the cancer is a hematological cancer, preferably AK,.
As used herein, "hematological cancer" refers to a cancer of the blood, and includes leukemia, lymphoma and myeloma among others. "Leukemia" refers to a cancer of the blood, in which too many white blood cells that are ineffective in fighting infection are made, thus crowding out the other parts that make up the blood, such as platelets and red blood cells. It is understood that cases of leukemia are classified as acute or chronic. Certain forms of leukemia may be, by way of example, acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL);
chronic myelogenous leukemia (CML); Myeloproliferative disorder/neoplasm (MPDS);
and myelodysplastic syndrome. "Lymphoma" may refer to a Hodgkin's lymphoma, both indolent and aggressive non-Hodgkin's lymphoma, Burkill's lymphoma, and follicular lymphoma (small cell and large cell), among others. Myeloma may refer to multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain or Bence-Jones myeloma.
In some embodiments, the method further comprises screening the population of cells for a NPM1c mutation and wherein the cells in the population do not comprise a NPM1c mutation.
In an aspect, there is provided a method of cancer therapy or cancer prevention in a patient, the method comprising providing a treatment to the patient that at least partially eliminates cells containing at least one of a DNMT3a or IDH2 mutation. In other aspects, the treatment could also reduce/eliminate the cancerous properties of such cells, and/or reduce/eliminate their potential to become cancerous.
Such treatments include therapeutic candidates that target cells containing such DNMT3a or IDH2 mutation. One example of a therapeutic candidate being developed to target IDH2 mutation containing cells is AG-221 by Agios Pharmaceuticals.
In some embodiments, the method further comprises, prior to treatment, obtaining a sample from the patient, screening the sample for cells containing a DNMT3a or mutation, and determining the existence of the DNMT3a or IDH2 mutation in the cells.
In some embodiments, the patient has previously received chemotherapy and/or radiation therapy.
In some embodiments, cells in the sample do not contain a NPM1c mutation.
DOCSTOR:293511613 In an aspect, there is provided a method of assessing a patient for risk of cancer, the method comprising: screening cells from a patient sample for mutations in DNMT3a or 1DH2; and determining a patient is at risk of cancer if the sample contains cells containing the DNMT3a or IDH2 mutation.
In some embodiments, the risk of cancer is the risk of cancer recurrence following cancer therapy.
In an aspect, there is provided a method of monitoring cancer therapy in a patient, the method comprising: screening cells from a patient sample for mutations in DNMT3a or IDH2; and determining an effectiveness or progression of therapy, wherein effectiveness or progression is directly related to a decrease in cells in the sample containing the ONMT3a or IDH2 mutation.
Optionally, in any of the foregoing, the cells or population of cells comprise at least one of hematopoietic stem cells (HSC), megakaryocytic-erythroid progenitors (MEP), multilymphoid progenitors (MLP), common myeloid progenitors (CMP), granulocyte monocyte progenitors (GMP) and mature lymphoid cells.
DNMT3a mutations and IDH2 mutations that have been characterized as being associated with cancer have been described in the art.
Optionally, in any of the foregoing, the DNMT3a mutation is the R882H mutation or the RI 37C mutation.
Optionally, in any of the foregoing, the IDH2 mutation is the R1400 mutation.
Optionally, in any of the foregoing, the DNMT3a or IDH2 mutation results in loss in function of DNMT3a or IDH2, respectively.
In an aspect, there is provided a composition for use in cancer therapy or cancer prevention, the composition comprising a compound that at least partially eliminates cells containing at least one of a DNMT3a or 101-12 mutation, along with a pharmaceutically acceptable carrier.
As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
Examples DOCSTOR: 2935116\3 of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.
The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention.
EXAMPLES
Methods and Materials All patient samples listed in Tables 1, 3 were obtained under Research Ethics Board approval with informed consent. Non-leukemic stem, progenitor and mature cells were sorted from diagnostic samples and subjected to genomic analysis. 11lumina sequencing libraries were constructed and target enrichment was performed using a custom Agilent SureSelect kit (following manufacturer's protocol). Sequencing was conducted on the 11lumina HiSeq 2000 platform to an average on target coverage of 250x. Reads were aligned to the reference human genome build hg19 using Novoalign (Novocraft Inc.), and a BAM file was produced for each tumor and T-cell pair. Variant calls were made using the genome analysis tool kit (CAN.
Significance levels (P values) were determined by chi-square test. Targeted Sanger sequencing and ddPCR were performed for specific point mutations. Primary AML samples were also transplanted in xenograft assays using standard conditions.
Targeted sequencing of leukemia-associated genes. Genomic DNA (gDNA) was subjected to limited whole genome amplification (RepliG, Qiagen) to obtain the required amount of input DNA for the SureSelect protocol, Amplified gDNA was mechanically sheared using the Covaris M220 Focused-ultrasonicator, and Illumine sequencing adaptors were ligated to fragments to make a sequencing library, which was then hybridized with 120mer biotinylated RNA library baits to capture the regions of interest. Baits were designed to capture the coding sequence of the 103 leukemia-DOCSTOR: 293511\3 associated genes (total target size ¨370 Kb). We initiated the generation of the HALT
Pan-Leukemia Gene Panel in mid-2012. To compile the gene list, we collected information from three sources: 1) Literature - we screened publications describing large-scale mutation screens in different leukemia subtypes; 2) Mutation Databases -6 we screened databases from the International Cancer Genome Consortium (ICGC:
http://dcc.icgc.org), the Cancer Gene Consensus (http://cancer.sanger.ac.uk/cancergenome/projects/census/), and the Catalogue of Cancer Gene Consortium (COSMIC;
http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/) for somatic mutations reported in AML, MDS, B-ALL, T-ALL, CML, and CLL.; 3) Leukemia Experts - we asked HALT investigators to submit genes of interest in leukemia, including MDS/AML, ALL, CML, and CLL. We curated the fists for non-synonymous mutations that can be analyzed by targeted exome sequencing (leading to the removal of genes that are uniquely associated with transIocations, inversions, complex rearrangements and copy number alterations). We further removed genes in the COSMIC and ICGC lists that were observed in less than 2% of samples, except for genes recommended by a leukemia expert. The final list of 103 genes was reviewed and agreed upon by consensus. The targeted regions were pulled out using magnetic streptavidin beads and amplified. The resulting amplified library was quantified and sequenced on the Illumine HiSeq 2000 platform to an average on target coverage of 250x. Reads were aligned to the reference human genome build hg19 using Novoalign (Novocraft Inc.) and on-target single nucleotide variants (SNVs) and insertions and deletions (indels) were called using the genome analysis tool kit (GATK). Somatic SNVs were called in AML blasts with a read depth of at least 30x.
T cell isolation and expansion from primary AML samples. CD3+ cells were isolated from peripheral blood (PB) AML patient samples using EasySep (Stem Cell Technologies) and re-suspended at a concentration of 1x107 cells/2 mL in RPM1 +
10% FBS-HI + rhIL-2 (250 IU/mL, Proleukin, Chiron) + anti-CD28 antibody (5 pg/mL, clone CD28.2, eBioscience). Cells were then added to one well of a 24-well plate that had been pre-coated for 2 hours with anti-CD3 antibody (Clone OKT3, eBioscience) and cultured for 4 days at 37 C with 5% CO2. Cells were harvested on day 4, resuspended in fresh RPMI + 10% FBS-HI + rhIL2 (250 1U/m1) and replated into one well of a 6-well plate. Cells were further cultured and expanded for 14-20 days, feeding with fresh full medium containing rhIL-2 (250 [Uhl) every 3-4 days. At the end of T cell DOCSTOR: 293511613 13 expansion, the purity of CO3+ T cells was checked by flow cytometry. DNA from the cultured T cells was extracted by PureGene Cell kit (Qiagen).
Droplet digital PCR (ddPCR). Genomic DNA (25ng) or amplified DNA (2 pl from a 1:20 dilution of a 16 happlicants' RepliG whole genome amplification) was subjected to ddPCR in a 96-well plate according to the manufacturer's protocol. Each sample was tested in duplicate. The plate was then loaded onto a droplet reader with a two color FAMNIC fluorescence detector. The mutant allele frequency was calculated as the fraction of positive droplets divided by total droplets containing a target.
To evaluate the detection limits of the ddPCR assay, a standard curve was generated using serial dilutions of DNA with a known mutation frequency mixed with non-mutated DNA.
The minimum detection level was 1:1000 (0.1%).
Fluorescence activated cell sorting of human stem/progenitor and mature cell populations. Mononuclear cells (1x106/100 pl) from peripheral blood or bone marrow of AML patients were stained with the following antibodies (all from BD unless stated otherwise, dilution used and catalogue number in parentheses): anti-CD45RA-FITC
(1:25, 555488), anti-0090-APC (1:50, 561971), anti-CD135-Biotin (1:10, 624008), anti-CD38-PE-Cy7 (1:200, 335790), anti-CD10-Alexa-700 (1:10, 624040), anti-CD7-Pacific Blue (1:50, 642916), anti-CD45-V500 (1:200, 560777), anti-CD34-APC-Cy7 (1:100, custom made by BD, CD34 clone 581), anti-CD34-PerCP-Efluor 710 (1:100, e-Bioscience 46-0344-42), anti-CD33-PE-Cy5 (1:100, Beckman Coulter PNIM26471.1), anti-CD19-PE (1:200, 349204), anti-CD3-FITC (1:100, 349201), anti-CD56-Alexafluor 647 (1:100, 557711), and Streptavidin-QD605 (1:200, Invitrogen 010101MP).
Samples from Patients #1, 10, 11 (remission sample only), 32, 35, and 55 were enriched for C034+ cells using a Miltenyi CD34 MicroBead kit according to the manufacturer's protocol prior to antibody staining. Cells were sorted on a FAGS Arialll to a post-sort purity of >95%.
Xenotransplantation assays. Animal experiments were performed in accordance with institutional guidelines approved by the UHN Animal Care Committee. 8 to week-old female NOD/SCID/IL-2Rgc-null (NSG) mice were sublethally irradiated (225 cGy) 6-24 hours before transplantation. Mononuclear cells from AML patients were depleted of CD3+ cells by EasySep (Stem Cell Technologies) prior to intrafemoral transplantation, Mice were sacrificed 8 or 16 weeks after transplantation and human engraftment in the injected femur and non-injected bone marrow was evaluated by DOCSTOR: 935116\3 14 flow cytometry using the following human-specific antibodies (all used at 1:200, all from BD unless stated otherwise, catalogue number in parentheses): anti-CD45-APC
(340943), anti-CD19-FE, anti-CD33-PE-Cy5, anti-CD3-FITC, anti-CD14-PE Texas Red (Beckman Coulter PNIM2707U), anti-CD15-Pacific Blue (642917), anti-CD38-PE-Cy7, and anti-CD34-APC-Cy7, The threshold for detection of human engraftment was 0.1% CD45+ cells. All flow cytometric analysis was performed on the LSRII (BD
Biosciences). For limiting dilution assays, the frequency of repopulating cells was calculated using ELDA software/2.
Statistical analysis. For the initial targeted sequencing analysis, 12 independent patient samples were studied to capture the biologic diversity of AML. For validation of the DNMT3a findings, 71 samples were screened in order to identify at least 15 with DNMT3a mutations, as predicted by the known prevalence of DNMT3a mutation in AML. For limiting dilution analyses, at least 25 xenografts were analyzed for each patient sample to ensure a large enough sample for statistical comparison. No animals or samples were excluded from any analysis. No formal randomization method was applied when assigning animals to different experimental groups. Group allocation and outcome assessment was not done in a blinded manner, including for animal studies.
Frequency estimations were generated using the ELDA software, which takes into account whether the assumptions for LDA are met (http://bioinf,wehi.edu.au/software/elda/index.html, provided by the Walter and Eliza Hall Institute)49. P-values were derived using two-tailed Student's t-tests.
In each group of data, estimate variation was taken into account and is indicated as standard deviation. For all graphs, * p=0.01-0.05, p,--0.001-0,01, and p<0.001.
Results and Discussion During studies to examine intra-tumoral genetic heterogeneity in AML, deep targeted sequencing (Tar-seq, read depth ¨250x) of 103 commonly mutated leukemia genes (Fig. la) was carried out on peripheral blood (PB) samples from 12 patients at diagnosis (blasts >80%)(Table 1, 2). Normal T-cells were expanded in vitro to provide non-leukemic tissue for genetic comparison. Consistent with mutant allele frequencies reported in recent studies a DNMT3am' was found in 4 of 12 samples (mutant allele frequency ¨50%) (Fig. la). Unexpectedly, in 3 of these 4 patients, DNMT3a' was DOC$TOR: 2935116\3 15 detected in T-cells at a low allele frequency (1-20%). Other genetic alterations including NPM1c were found only in PB but not T-cell samples, ruling out AML
cell contamination of cultured T-cells. To estimate the proportion of AML cases with DNMT3e&bearing T-cells, 71 additional samples, taken at diagnosis from patients with normal oytogenetics, were screened by Sanger sequencing for DNMT3amut along with other common AML mutations (Table 3). Consistent with published datau'l, 17 of 71 AML samples (24%) carried mutations in DNMT3a, and 15 of these 17(88%) also carried NPM1c. For these 17 patients, the allele frequency of DNMT3dnut and NPM1c in GD33+ blasts, as well as corresponding freshly isolated T-cell controls, was measured by droplet digital PGR (ddPGR) at a sensitivity of 1 mutated allele in 1000 reference alleles. Whereas both DNMT3a`nuf and NPM1c were always present in blasts at similar allele frequency, DNMT3amut with no evidence of NPM1c was detected in T-cells from 12 of these 17 patients (70.5%) (Fig. 1b). In addition, FLT3-ITD
was detected in blasts but not the T-cells of 2 patients bearing this mutation (Fig. 5), These data reveal the sequential order of mutation acquisition in these patients, with DNMT3ainut arising earlier in leukemogenesis than NPM1c and FLT3-ITD, a conclusion predicted from recent studies on bulk AML blasts showing that NPM1c and FLT3-ITD
occur late and are the only genes recurrently mutated in DNMT3am11t AmL15.28,32.
Moreover, applicants' findings establish that DNMT3amt occurs in an ancestral cell that gives rise to both T-cells and the dominant AML clone present at diagnosis.
To gain insight into the properties of the ancestral cell within which DNMT3dr" first arises, applicants examined additional non-leukemic hematopoietic cell populations from 11 DNM13amt/NPM1c AML patients. A high resolution 12-parameter sorting strategy33-5 was employed to isolate non-leukemic hematopoietic stem and progenitor populations, including hematopoietic stem cells/multipotent progenitors (HSG/MPP), multilymphoid progenitors (MLP), common myeloid progenitors (CMP), granulocyte monocyte progenitors (GMP), and megakaryocyte erythroid progenitors (MEP), as well as mature B, T and natural killer (NK) cells within the CD33¨ cell fraction.
Together with CD45dimCD33+ AML blasts, these highly purified, phenotypically defined normal cell populations were assessed by ddPCR for DNMT3amut and NPM1c (Figs. 2, 6, 7).
DNMT3amus was found together with NPM1c in C033 blasts from all patients. By contrast, applicants found DNMT3amt at variable allele frequency without NPM1c across the spectrum of mature and progenitor cell populations. Results for a representative patient (#11) are shown in Fig. 2a-b. In this patient, DNMT3an't was Docs-roR: 293511 (5\3 present in HSC/MPP at an allele frequency of 12-30% without detectable NPM1c.
Although the clonal contribution that an individual normal HSC makes in humans is unknown, studies in higher primates estimated that single HSC provide approximately 0.6% clonal contribution during steady-state hematopoiesis. Thus, the high DN/V/T3amut allele frequency in HSC/MPP points to their clonal expansion as compared to non-mutated FISC. In Patient #11, FAMT3a1t was also present in all downstream progenitors at variable frequencies. The mean allele frequency among HSC/MPP, MLP and CMP was 24.6% across all patients analyzed (Fig. 6). Importantly, even for patients in whom DNMT3e1' was not detected in mature cells, DNMT3amut without NPM1 c was found in stem/progenitor populations (Fig. 2c), providing further strong evidence that DNMT3amut precedes NPAtic during leukemogenesis. Interestingly, in 3 patients (#10, 14, 16), DNAAT3am1t was detected in CMP but not HSC/MPP, an outcome consistent with the existence of DNMT3eut-bearing HSC below our detection limit that generated a clonally expanded CMP population, or possibly the existence of a preceding lesion in HSC/MPP with later acquisition of DNMT3amu in CMP.
Resolution of this question will require whole genome sequencing of sorted blast and phenotypically normal populations from patient samples. Applicants' analysis also showed that in 6 of 11 patients, both DNMT3a"' and NPM1c were found together in MLP and/or GMP populations, pointing to the likely progenitor cell types where overt AML driven by NPM1 c arises. Collectively, applicants' findings provide key insights into the leukemogenic process in human AML and confirm historical predictions from early cionality studies of the existence of a pre-leukemic state3'37.
To examine how DNMT3amut affect population dynamics during leukemic progression, applicants undertook temporal analysis of mature and progenitor cells from 5 patients (#11, 28, 35, 55, 57) sampled at diagnosis, remission (3 months) or relapse (Fig. 2b, d, 7, 8). Compared to diagnosis, the allele frequency of DNMT3ang alone was similar or higher at remission (Patients #11, 28, 35, 57) and relapse (Patient #11, 55).
Although CD33+ leukemic blasts at diagnosis always carried both mutations, CD33+
myeloid cells at remission bore only DNMT3ed, suggesting that they are not AML blasts but the progeny of DNMT3amut-bearing progenitors with preserved myeloid differentiation capacity. In the relapse sample of Patient #11, both mutations were present in the majority of cells, with the exception of 1-ISUMPP in which a proportion carried only DNMT3amut. Patient #57 was a long-surviving patient that allowed a comparison of early and late (36 months) remission samples and showed a striking increase in DOCSTOR; 2935116\3 17 DNMT38m' allele frequency in most cell populations over time (Fig, 2d). In addition, a small proportion of CD33 myeloid cells in the late remission PB sample contained both DNMT3amlit and the NPM1c mutation found at diagnosis, suggesting either regrowth of the diagnostic leukemic clone or emergence of a new clone following an independent NPMic mutation event within the pre-leukemic pool. Collectively, applicants' data indicate that the ancestral cell that bears DNMT3e1 without is an HSC/MPP capable of multilineage differentiation. Moreover, these ancestral HSC/MPP survive chemotherapy, expand during remission, and might serve as a reservoir for clonal evolution leading to recurrent disease.
To establish conclusively whether phenotypically defined DNMT3amut-bearing HSC/MPPs are functional HSC and whether competitive repopulation advantage underlies their in vivo clonal expansion, applicants undertook xenograft repopulation assays. Mononuclear cells from the PB of 2 patients at diagnosis (#11, 55) with DNMr3en't allele frequency in HSC/MPP of 30% and 20% respectively were transplanted into cohorts of immune-deficient mice using a limiting dilution approach and analyzed after 8 and 16 weeks (Fig. 3, 9). In this xenograft model, leukemic engraftment is characteristically seen as a dominant myeloid (CD46dimCD33+) graft, whereas non-leukemic grafts are multilineage and contain both lymphoid (predominantly CD19+ B cells) and myeloid (CD33+) cells (Fig. 10). For Patient #111 multilineage engraftment was seen in 24 of 36 mice, giving a calculated frequency of 1 repopulating HSC in 7.3x105 cells (Fig. 9a). Only a single graft contained more than 60% CD33+ myeloid cells, suggesting co-engraftment by a leukemia stem cell (LSC) that was present at low frequency. Applicants analyzed by ddPCR 12 of the multilineage xenografts following 16 weeks of repopulation, Ten of these contained a high proportion of cells bearing DNMT3amul without NPM1c (mean allele frequency 57%), whereas both DNMT3amig and NPM1c were present in the single mouse with significant myeloid engraftment (Fig. 3b). Kinetic analysis demonstrated increasing DNMT3an't allele frequency in multilineage grafts over time (Hg 30). Similar results were found for Patient #55 (Hg. 9 and data not shown). In contrast, cells from the relapse sample of both patients generated leukemic grafts and no multilineage grafts (Fig. 3a and data not shown), consistent with a higher LSC frequency at relapse compared to diagnosis. Together, these data provide evidence that DNMT3arnui occurs in HSC/MPP capable of generating a long-term multilineage lympho-myeloid graft, confirming their designation as pre-leukemic HSC 2--Q (preL-HSC).
DNMT3aw also DOCST0R: 293511613 I ft endows preL-HSC with a competitive repopulation advantage over non-mutated HSC

explaining the clonal expansion of preL-HSC in patients at the time of diagnosis and during remission.
Applicants' xenograft results indicate that when preL-HSC exists at higher frequency than LSC, non-leukemic multilineage grafts, rather than leukemic grafts, are frequently generated, xamination of applicants' historical xenograft data from 264 diagnostic AML samples revealed that 37% did not generate any graft, 40% generated leukemia, and 23% gave rise to non-leukemic multilineage grafts (Fig. 10). Sanger sequencing data was available for 26 samples that generated multilineage grafts (Table 3) revealing that 10 of 25 (40%) came from patients bearing DNMT3arnut; 1DH1/2 mutations were present in 12 patients, including 3 who had both DNMT3a and mutations (Fig. 4a, 4b). To examine whether pre-leukemic cells also exist in patients with IDHI/2 mutations, applicants analyzed samples from 3 patients with IDH1 and 3 patients with 1DH2 mutation by high resolution cell sorting and ddPCR. In 4 patients, no pattern of preceding mutation was detected in non-leukemic cell populations.
However, in 2 patients applicants found 1DH2 mutation without NPM1c in a number of progenitor and mature populations (Fig. 4c), suggesting that 1DH2 mutation might also occur as a pre-leukemic event Applicants' data predict that DNMT3a mutation may occur in healthy adults and pre-date AML diagnosis by months or even years. Through searches of exome sequence databases derived from PB (httos://eso.ds.washinoton.edu/druoal/) applicants found that the frequency of the DNMT3a R882H variant (rs147001633) was 0.066% (3 in 4545). Although this was considered to be a germline variant in this healthy adult cohort, applicants' findings raise the possibility that the mutations detected in these studies may have originated from an HSC/MPP containing an acquired somatic DNMT3a mutation that underwent clonal expansion.
Applicants' study provides a number of key insights into the leukemogenic process in human AML Applicants' findings establish the sequential order of mutation acquisition for the patients reported here: DNMT3a' occurs before NPM1c and FLTI-ITD.
Additionally, applicants provide strong evidence for the presence, at diagnosis, of preL-FISCs that are ancestral to the dominant AML clone. Based on applicants' data, preL-HSC are prevalent among patients with DNMT3a', which account for 25% of adult AML cases; additionally, applicants' multilineage engraftment data suggest that DOCSTOR: 2935116\3 preL-HSC may also exist in a proportion of AML patients with IDH2 mutations.
Pre-leukemic progenitors of varying phenotypes have been reported in other types of hematologic malignancies25'g41, although functional studies were limited.
Applicants' work supports prior studies identifying phenotypic primitive cells that bear only a subset of mutations found in AML blasts26'27=42. The more precise analysis of highly resolved HSC and progenitor populations that applicants have undertaken provides novel insight into the identity and proportional contribution of the stem/progenitor populations that acquire pre-leukemic lesions. Furthermore, applicants' work demonstrates that DNMT3amut confers a functional repopulation advantage to preL-HSC over wild type HSC in xenograft assays, which likely underlies the clonal expansion of preL-HSC observed in patients at the time of diagnosis.
Applicants' study is consistent with mouse studies showing that HSC lacking DNMT3a have a competitive growth advantage43'44, and with a recent report predicting that the human DNMT3amur results in loss of function42, 16 Collectively, applicants' results support a model wherein the cell of origin for DNMT3am`l AML is an HSC and the initiating DNMT3a mutation results in the generation of an expanded pool of HSC and downstream progenitors, within which additional mutations such as NPM1c are acquired, driving progression to AML.
In the samples studied here, applicants' findings point to GMP and/or MLP as the likely populations in which NPM1c was acquired, Applicants' results have broad clinical implications. Prior studies in T-ALL
and B-ALL7=11.14 revealed the existence of genetically diverse subclones at diagnosis. As found originally in these diseases lz and now in AML, in approximately 50% of patients the relapse clone is not related to the predominant clone at diagnosis but rather to a minor leukemic subclone 1 ./5 or to a predicted ancestral clone'l. Applicants' direct demonstration that ancestral clones persist at remission suggests that preL-HSCs are resistant to induction chemotherapy and for some patients they might represent a reservoir from which relapse arises, PreL-HSC should be directly targeted to prevent relapse. Drugs that effectively target mutations in DNMT3a or IDH2 (e.g. AG-221) that give rise to preL-HSC, may be an opportunity to eradicate these preL-HSC
clones before the acquisition of additional mutations renders them more resistant to therapy.
Applicants' findings also support broadening the definition of minimal residual disease to include not only the post-therapy survival of AML blasts and LSCs but also preL-HSC. Practically, this suggests that for patients with both DNMT3amul and NPM1c, the DOCSTOR: 293511613 70 residual level of both mutations and not NPMic alone should be monitored.
Finally, applicants' database analysis showing that the DNMT3a ii882H variant is present in blood samples from normal adults suggests the ability to determine the risk of progression to AML; enabling earlier diagnosis for those patients who present without prior overt hematologic disturbances.
Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein are incorporated by reference.
DOCSTOR; 2935116\3 Table 'I
. FLT3-ITD Induction Response ___________________________________________ .
tt ! ABIVIT ; DONOR Status at time of Status at last ;
!
=
.

. Consolidations i I
transplant ! foiiow up i 1 , .
, =
1 , = , .
, [I , rid _ 7+3 complete remission 1 2 yes ! related ! CR1 f _ dead rid 7+3 F no response . 0 1no ;
dead rid 7+3 n 4..
o response i 0 no : 1 dead high 7+3 complete remission 1 2 no i I o . . -I. -=--- -----¨ - --- i dead _ rid _ , 7+3.. com -- - plete remission I ! 2 no i dead 0 o _ . .......... .. ._ - . . ..
1 N.) intermediate 7+3 no response I / no = ?.
I
dead co Ø
rid 17+3induction-related death i 0 no , i dead N.) = -= 0, nd supportive 1 0 no _ . = dead w (xi intermediate 7+3 no response i 2 I no. E
dead N.) 1- _ .
..... .,.. 0 _ negative 7+3 complete remission ! 2 yes i unrelated ' CR1 : dead 1-, Ø
negative 7+3 complete remission I. _ . 2 no i )- - i dead i = i 1 N.) rid 7+3 complete remission ; 2 __ no 1 I alive i i 1-, 1-, DOCSTOR: 293511613 00000.000=000.000.g00 000000 .13 l0000 L,,,i00000000000q 0,0000c..VG.C00000 00.0 ' ..71noodooc.000 '21000 .-oc000000l,...Rlooacrocip , _E
.7.4 = '..1 õ7.7............o$0.....mool...v. .., z' ci a 'gloo:l000000p0000pooloo o'oo2 o 1.7 CI 1_ d d 6 =
. . , j .2 00 0000000000000000000000 1 T,0000000M0O000000.000 0000 i0..00.00000.000g0000000.0 I

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.. . 5

2-, or. it; . T.
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2.-= V, ..,, A
.3.00000000.010.00A:000.0000 00000000000000000000=000 Z00.0000000 000pR000riol00 O
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t 5 t -.
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.,i sr- .1.il - lz .L=J:1=gaMOrrilita, 41!^1 in7AN'AWVAr.:04 M,,jAr, lt"Ii4liggiUOT.AUe'09,1 8.D.sPil Itgat,T.TASSUVq1a2.1-1-1 N.,..7wZrErti5000.5,mVX_:-.3 F.,1,-., ¨I I- ;
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o- . . = - .. -- -= ,.. ... õ .., .. . = N
m s R +a u:.

c\I
tl:Ogyuci,..l.¾,WW.LIPU,-Ull.,,,<....1 1,O4u......(voLluO.u.,u1OVUrCuLk, M
N t g t 0 = = ' = = = = =
_ .:-.
'm = - -0 -=7.,-.P..IA
............................................................. 22- = =P -I
-,,.' g g i VV
? V 2 2V
W3nPV-322-1-c-a2r:ORP'M - -.. -..= ¨ .; z=st-.., ,. .., ifc fl.
cn n n ., gmlimr.'itgizl-Ftgimtil-mm¨Ig tl.3a',,I.OtwHm05aAAV .,,naiwAl¨?-na2 1.-nnl-eA0iTqggmggi..12AgCiRn.FA.'=.%
mOol.R2,14nr.5.9-wriax413Namm r.la..,ap4 ,,1,5-1.r,p,nnrn :IVnIRMFiPqAUmlAn8si E

g2g....¨Naww-4.,--4mgNa..-v-g:1 2.4.-4,,...." . n ** r.lm.....,p,gg...n .... nrA
a tAtIEHxzlzFA325.55MREEgM mAg ...*-*00.+m,.,N,-..N .-, ovvgam wre.1..
HUISMgSIMAE5,T,Z1 N

,e) 25 1 -, ra rn .* ,.. ., . s.g :1 a ':-,3 M:-'1 Y1 :',- !I :1 Va' ;4' .1 :2 ;.1 n A '1.-..4r.ym.,,...9:4f1:24r.F4742r4 =,.,--,-.w.-..9:11o.,4:-.4.!..2.7;g14P.n.T.
C-) al V

i¨ , A

p.m!. 7 Pettent 9 s5NV 24ne Chownommom PM , ID REF ALT , &don , _ MAL 7 teas ANIL Thus 1 3541.1 10 5024005 15162191716 El A synorryinouve dart 0 4 0 ' 0 2 . ATM 11 .109206514 . A G Asp272351v 0 0 0.481599 ' 0

3 FICOR X 59934139 = . C , . A 01y1547 a µ55103: 6 113641949 - , . C T Glv64195er 0 ' 0 , DNMT34 2 . 25457242 i15147071699 C 7 Ar6602H5 . 0 0 0 ' 0 6 ONIff94 2 25470498 ! . 5 A 4/518Cri . Or 7 07312 7 1.112.06775 . Tr T rfaMeihift..yi Ha nt . 0 0 , P,771,194 0 9 IONS 15 90631934 ' r512191350Z C T AMADGIA 0 .
0 0 o .
, 1.0 tit a 85509661 . 6 GCTC Trp557delln36361er . o o , o o 11 . XR 4 55593007 = A G 49558411 0608. 0 11 4648 . 12 25993234 tre121913529 C 51012930 I 0.06444 , 0 0 , 0 .
la . NFM1 5 . 170237547, - . 6 GTCTG
framesNitryartant . 0 0 0 , 0 14 NRAS 1 115256930 10121313254 00 514674p a a o o leltra 1 115239744, r3121434596 C 7 51y134sp . 0 0 16 651101 , 21 36206437 . . 0 I3FCF
Leu215,..340.1E4nsMa 0 0 0 , 0 17 RU1101 , 21 26732854 '. . C . r wyma. , 0 . 0 0.633333, 0 .
16 RUNX1 21 56206836 . . T G. . 855226442 0 0 0 /9 59391 . 2 01267391! . C A Lys666301 0 0 0 ', Q
52103 10 .112349703,, . MOO A 0644661 . 0 0 0 , , 0 21 7E72 4 106157329! . c T ormax o . o o 0 .
22 . 7572 ' A .1091973i5, . C CC
trameshlir_earlant 0 0 0.008 0 23 TET2 4 .10615-09121 . C 7 , . kr1261Cy1 0 0 0 ; 0 Id TE71 4 1061649281 . A _ C . downstreanwen,variaM , 0 0 0 . 0 .
WT1 11 . 32417279 . . C 04 414961411v 0 0 o , o 26 Wit 11 52117914 . . GTACAAGAGTCG G a prIme_UTR_varlant,NM_Lransellpt_erMant 0 0 0 0 . , 9151050 Pollens 10 r5NV Gene Chromosome 905 117 REF 447 Code. AML
7 celh AML 7 selh 2 MAI 20 31024026 :. n150391710 a svnenyrnouLya Pa nt 0 o o o 2 ATM 12 05106504' , A 0 A1171272501i a o o o 3 13105 X 395341.39 . C0 Gly15.17 0 0 o 0

4 11I1109 6 219341949 . C 7 00y6095er a a o o i 06507 15 , 2 25427202:10147001w I I ' Ar461,1141. 0.145132 0,136667 0.576033 0 . .
6 DNNIT37 2 254704913 . G A Ar5326C7s. 0 . 0 0 , 0 7 . 12502 7 148504775. . . Ti T 01 fttOd rf,....vi MOM . o o o 0 9 10561 25 90191114 . 5421113502 C T 564148310 0 10 . KR 4 550936514 . 0 0C7C : T925.5744 11.nsio 0 0 , 0 0 11 101 4 55593607 ,, . A G 1.71554146 o o o o , 12 100.1 12 , 25398284 ' n121113529 C T 61y1.24sp 0 13 11050I S .00531147 . 4 41'C134 ' f mil erhitrident 0,5 0 0.46 0 14 NAAS 1 123256530, 10121911214 G T G1n611ys 0 0 0 , 0 15 NRAS 1 1.10136744'15121154590 C T , Gly1.9346 a o 0,22115/ 0 10 94501 . 21 :35256007 , . 4 0034 .
Le1223,_,Ser2241164.4 0 , 0 0 0 17 RUN= 21. 36252854': . C 7 5041704 o o o o -15 509 01 22 16206535 I . T 4 ' Ser210Aie , 0 0 o o 19 173131 2 138267259 . . C A : 1.74666650 0 0 o =
10 SMC9 xa , 02345705 . AAGA A , 41114469041 0,963 0 0 . 0 21 7E72 4 '104152329; . C r 5157440 0o 0 a . ' 22 TET2 4 1061573891 . C CC ' I ramesNivarlant , 0 0.061 0 0 19 Ten 4 06144013 . C r 438114449 0 0 , 24 TET2 4 105161923 . A ., c 40wrstre4r1,5ene,7,ariant 0 .. o 0 , 0 25 WI'! 11 32417909 , . õ C CS ' .1.16367.62y 0 0 0 0 16 ViTi. 11 92117114 . . , GTACAACIAGTCG 0 ' 9.6tiate_UTre_ve4ire,N515.6.4escrlavre56e04 0 0 fl 0 14564111 9.661111 drili , Gans . Chrocno Lamm 036 . lb , AC; . ALT .
Codas AML YO04 AML Tcelk synonymous,..eadont o o a o 2 ATM 11 1082065941 . A a Asp.272321v. a e 0 ' 0 .
3 151911 , X 39934139; . c A Gq254T 0 o o a 4 CSMO3 it 113841349, . C 7 5174095e5 0 , 0 a 0.604062 =

5 ,51034154 2 . 254572431 n147012555. C I k-6661Hty Ø175992 0,0743 0 0

6 ONNTIla . 2 23179493: . G A Ars,316C0s o 0 0.457711,0.011976

7 . E2511 7 544504175. . . 77 1' Da mretarviehas . . 0 0 0 ' 0 9 1042 13 90631934 15121913502 C 7 0681.40515 0 0 6.514694 ' 0.0145E3 , 20 an 4 55593604 '. . 6 GC711 T1o5574e1r51C0s3er 0 0 6.251555 o 11 . %It = 5015340 ' . . A 0 , Lyrad9Arg 0 0 0.35MC0 0 12 MIAS 12 25398184 118121913529 C T GN1293p a 0 11 944.1.41 1 176637547. . G 51070 from..hilviehnt . 0,40 0.2 0 0.004 14 N745 1 125250530 n022913254 0 T 01401551 0 0 o .0004149 15 . rims 1 115156744c rs11143A596. C T 5171340p 0.004016 0 o o 16 RUNX1 21 56105637 . . a GGC61 Le6225jei22EforAIA 0 0 a o 17 RUNY1 21 56251154 , . C 1 3ly170.4, a 0 0 o 19 , MI1121 21 _ 36205126 , . , y 0 5e1226930.
0 o 0 a 19 5F361 2 198267359 . c A Lvs666Asn 0 0 o o 20 .. SMC3 10 .111940703 . . AAGA A 1356455441 21 7511 4 106157328 . C T 016714 0 0 5 22 TET2 4 106157389 . C CC tranieshievariant , 0.004 0,008 0 0 21 7571 d .105164015: . . C T 6047.2400 , 4107403 o o o 24 TET2 4 106164929! ,. A c stmenstreamaene_mrlem o o o o 25 WTI 11 92417469 . . C CF 818982617, .
0 0 0 ' 0 15 ' wrz 21 524050 , . GI00405A57%5 9 2 prime OA vArlant,N30 transcr9m_varlant .0 0 0 0 Table 3 on following page.
DOCSTOR.: 2935116\3 ____________________________________________________________ -de novo vs. secondary V611CC it BM blast 26 at Patlent N Sex Age at diagnosis ;
fA5 Cytogenelics AML diagnosis ellagnOSIS
., _________________________________________________________ 13 64 45.9 : de nova i=Al , . 198 90 46,XY
.. . , la F 41.5 de nova M2 22 70 46,82 15 M 64 de novo Mi. . 357 90 46,X5 16 M 69.7 i de nOVO r.14 102.5 95 46)(Y
t F 17 41,9 de nova M2 j 41.2 00 46)3( .
99 BO 46,XY
, ; I@ M ' 36.9 = de novo M6o , ..
19 F , 44.4 =
, de novo M2 , 55.9 = 90 46)0( 20 F 56.3 de novo . NI4 ; 46.9 = 40 . .
21 j F ; 55.1 de novo M4 55 $0 . 1 . .
, 22 F 59.6 secondary (radiati0a) , Ml 4 az.s i 50 46,70t , 23 . F 74.0 = de nova 9=6 urciassified 75.9 . , 24 M 99 . de nova MI 4$
I CO 46,82 23 M 68.3 ' do.mvo ,,,,, ,. N14 39 65 45 xr .
....,..
26 F . 21.9 ' de nova M2 20.5 = 00 46,85 27 64 75 ' seconder/(rv3) nd i 6 nd .
46,82 26 F 49.7 de nova M1 , 104 act 46,}01 . , .
90 M , 61.8 do nova M1 . 204 150 .1-581' 31 j 60 36.4. de nova . MI 99 90 46.10' 82 M 61.7 de nova unclassified 145 39 95,XY
, i 02 F 21.1 , de novo unclassified . 29 70 46,XX
34 M 67.7 de nova MI . 121 : 99 96,10( . . .
SS F A0.6 de nova M5a SO 80 46...NX
, . .
36 ; M 93,5 secondary (rodlation) M4 ' . , 46,82 . . ' 37 r 35.7 . de nova uhelefelfied j 46 , nd 46,85 OS M 59 ; . de novo 114 25 25 46.XY
i 39 ' F 61.9 de Nam M5a 59 1 90 46,XX
. .
, =90 F . 27.3 .
de nova M2 150 j nd 45,85 , =
41 M 73.2 de novo Ala = 20 80 46,81' . . .=
' 92 F 66.3 ; secondary (chema) ; M5a 61 ' 90 46,28 49 . M ' 69.5 eecondary (MD5) nd 15 , 20 46,86 44 . M I 32.3 ; Rued lineage leakernlo .
mixed llnealie leukemia 51 90 4686 = as M 69.2 de novo M5a 89 0? 46.86 . .
' 46 F . 67.1 de nova unclassified 202 95 =
46,82 - = =
47 F = 693 de novo MI , 09.9 9-0 45,XX
95 F ' 65.7 , secondary (chemotrads) nd 99 60 46)0( ' , 49 M ' 60.4 secondary (radladon) MI 40 93 45,8e . =
50 DA 61.3 de nova nd 09 87 46,XY
.--........, -, -31 F 66 de 9060 MIS 29 20 48,85 52 F 70.3 de nova nd 11 nd 46,X4 , sa m ; 89.8 de nova M1 142 , 65 96, XY
, 54 . F , 613.7 , de nova M2 , 179 91 . , 55 Ivi , 75.5 , de nova MI , 60 90 46,XY
z .
56 F 75.6 ! de nova M2 = 15.0 , 22 46,85 , ' 57 j F 54.5 de novo MS. 44 94 46,58 ' 5111 5 90,9 , de nova M4 21 79 46,58.
.
59 M 715 i secondary (MIN) 602 32 60 46)0' ' 60 j F 59.1de novo. M1 ; ao , 40,85. -, , Si. M 65.2 , secondary fmrN) nd 62 52 46 XX
62 r 29.2 de nova unelseeined 10 nd . 46,86 63 M 423 ' de novo M5a 32 1. 16 46 XY
..
64 M 42.7 de nova M53 52 " 16 46,30' 65 . M 42.4 ' de nova MGa 2 1 as .
46,XY
. , , 1 , GO F j 61.1 ! de nova nd 523 ! 95 96,81' g7 M ' 62 ; de novo M1 4 - 26 90 46E. _ 65 -M 713.4 secondary (radiation) ; M4 a i nd 46,XY
ra r 44.5 ; . de nova nd 110 T, 35 . 46)0( 70 F ! 58.2 , de nova M5 33.6 ' 20 46,88 .
, .
71 1.1,,1õ , 59. ,. 516Ø3y9 . . M5a ! /67 90 46,XY
72 M 59.4 de nova ad 10/.3 95 48,82 73 F 61.7 ' de IWO rid 149.2 95 46,58 , , 74 . 9 94.$ , de 11020 M2 , 39.2 ;
Do 45,85 75 M - 79.8 ' Seco nds ry (rade don' AP3a 103 nd 48,8I
, r 76 . M . 84.2 . de hOVO M4, 20 20 45,8v 77 M 45.9 de novo mi 67 ' 90 46,XY
, 78 ; M = 72.9 i de nova MO 713 ' 90 .46,81' , 79 F 64.9 de novo Mb a 134 i 75 46,88 * . , so F .= 61.8 , de nova M1 334 , 95 40)5( Ell M , 76,2 secondary (radreelOn) nd 22 , 60 48,8?, , 32 M 723 ! secondary iradiationi M53 , 59 ' BS 46,XY
, BB F 70.9 de nova M4 63 rid n4 46,rf DOCSTOR: 2935116\3 9c _____________________________________________________________ ¨
sums at Sam et 1969P:09W
' Valiant I See MF.CCytopelletter Clair induction Response 0 Consolidellons Asmr Donor ABMT up (Ded 2012) , -; 13. M , , intermediate (normal) , 7+3 complete remission 2 , rsq . , dead id F Intermediate (normal) 743 eomprete remission 2 _ 19 , dead 15 m ; intermediate onmoou ' 741 , Induction=related death nodead . =
16 M Intermediate (normal) _ 743 , earnplere remission =
2 119 I , - dead 17 F kiterrned1403(ruari1all 743 complete mmission 2 riO*Ilya . =
, 113 ' M . Intermediate (normal) 743 , eamplete remission 3 you i Uimelette CR1 , alive /9 F .intermedIate !normal) 749 elovitTo Mmisslen 2 . yes . ' urrelated CR2 illIst 36 ' F Intermediate (normal) ,, 7+3 demplete remission . 2 no Olive = .., ; 21 , F Intermediate (normal) , 7+3 InduCtion-releted death . i .
, 22 F . 11R9M14S11Iil (normal) , 243 complete rerniMbh .
2Yes (sedated , CR1 dead . .
23 . F interned:are (normal) supportivedead . . .
241 M imprrnvdittp Irionmisli 7,3 . complete remission 2 no_ alive , 25 m imeanedotp (mama') 743 , complete remission 2 no.
dead 79 0 IMennediate (normal) 743 . mmplete r..I.1.1 2 no.
alive 27 M ' Intennediate (puma!) 7+3 oemplete remission 2 no dead 22 F , intermediate (normal) 7+3 corm:4m rom1991.. 2 .
're, unrelated CR2 try, coospiare remission 29 F Intermediate (normal) ', 7-3 2 , no aliot , 39 M Intermediate (normal) . 745 nO relpOnsib . 0 no dead . 31 M Intermediate irmonalI 742 Cgmellt. NMILIII611 2 no dead 32 nd Intermediate (normal) 743 complete remission 2 no alive 33 0 Intermediate (normal) 743 complete sernissioo 2 no dead 34 M intermediate (normal) . 743 no n99p4m4 6 no dead 35 F fmermedlate (normal) 74 complete ...minion no no alive 36 M intermediate Jnomtall clink:31111rd no dead ' 37 F intermediate !normal) 74,5 no relponae 1 no dead , 96 M Intennedlate (nonnal) 7+3 complete remisslon a . no alive 39 . F intermediate (normal) 7r3 complete remission , 1 , Yee . r,la tad C212 alive ' 49 . F Imenudialt (ronn9i) 7/3 complete remission 2 ' CB transplant . C.33 *Fut 41 M , intenedirrti (ramial) 743 . complete remission no deild '. 42 9 . 3orm94 99 (normal) 71-3 . complete remission 2 no aiNa 43 n4 . Intormeolimo Oornial} 7+3 . complete remission 2 no Rood =
44 M . Intermediate (nomial) 745 rCiftSpIMSE
alive 45 , M , Irtennediate (nOrrnal) 2.3 no response . 2 .. no dead 44 F Irtorrnediate (normal) 7+3 coincide remission 2 no dead .
27 _ F intermediate Invirniii) 743 eomplext remission 2 no ally, Ai F interned*" (normal) 7.3 no response dead 40 , a4 imowirwisinte (us mini) 743 , complete remission 2 no 630 . . .
56 ivl ; Intermediate (nOrm41) 743 , no resfoese.
1 I. dead . ' 51 6 kloro,00llo crg, nool) 7,3 , cam piece remlz Ion 2 no-aye , 52 F ; Intermediate (manna() 746 , eomplete remission 2 no arise 52 M Intermesh to (no noel) 743 , ee nlisl es*
r*MISSIOri 4 no a tue , 54 F IMermedlabe (normal) 7+3 complete retntition 4 . no dead 55 M Into-mediate (normal) 7+3 complete re mlbOon 3 ,Pcalive 56 F lot ormedlate (norrnal) clinical trial unknown 'is deed 57 , F intermediate (normal) 7,3 campfete remission , not clear , n? alive 56 F IMerrnediate(normal) 7+3 complete remission 2 _ no Ovid 53 M Intermediate (normal) supportive .
dead , 60 F . intermediate (hermal) 743 , complete remission 2 Yes unrelated CR1 dead - 61 M . imerrneritrte (normal) 74,3 . ee mpg te remission no 1 dead , 62 F . Interrosuila Os inorrobl( 7+3 0950 461* remission 2 , no , dead ' 63 IM Intermediate (normal) .743 . .. Lerripiete MMIOdeo 2 ; no olive , 64 M intermediate !normal) 743. e0M31letit remission 2 ! no alive 135 , M Intermediate [normal) 743 CoMplete ferninlen 2 , Yn ureilmitd DU dead ' 66 F Intermediate (normal) 7+3 , complete remIttlibh 1 . yu Pilibtd , CAI olive . 67 M Intermediate (norrnal) 74-3 . Cornplete leritlision . 2 no alive dB iri Intermediate (normal) 74-3 . Complete Mill-tile rr I no dead ' . 69 F Intermediate (normal) . 7+3 ; no response no &no , 70 F Intermediate (normal) . 7+3 complete rembrIon 4 iv:, alive 71 M . Intermediate (normal) . 7,3 ; unknovm no isliurt 72 M Intermediate (normal) 7+3 romciete remission 1 , yes related CR1 alive ' 79 F Intermediate (normai) . 7-3 complete remission nd no alive 74 . F intermediate (normal) 7+3 complete remission 2 yes related . CR1 alive 75 . M Inlyrootho to (6911.41) supporm. dead 70 IN Intermediate (rierrnai) ank4t Vial . no dead 77 M Into-modiste (normal) , 743 cemplete remission 2 yes unrebted CR1 alive 78 M . intMrriedlOte (,vprmal) 743 . nn respeme no dead , 70 F Intermediate (nermeg , 743 commetemmission no alive , , 60 F . intermedilte (rowno0 7+3 110 reSpOrge no dead , 61 M inteereirdiate (dermal) supportive no dead = = =
82 M intermediate internal) 749 complete remission nd no alive M F torermosiRma (cernsol) supportive no deocl DOCSTOR: 2935116\3 XonoroFt Patignt i Sex FL19.1TD
chr11:2160 602 4-21 08551 1F1.11-1103 5132a6 54.20927o7 NP911 chr5:1701037371.170637773 chwoctons-8.
____________________________________________________________ ---, , al , M multIlineage . + I.
'170537544-170137547 BeLdatfiC:14 1 =
,_ _ 14 F inulti8nes.416 + = ,,,, J70397544-170137547 htl_UTUTG
¨ic r - - Ni ' AML . + = .
=
16 Ni moldboard! + 1 :170137544-170137547 htt_thipTCTG
=
=
' 17 . F õ no graft, T - I .
11 . M no graft +.170837544-17003754 7 ha LchipTCP3' =
muitliine -age, no graft '' 20 F . no graft - 170137143_170 69714 4het_InsTG:CT ' , 21. , F multillneage . - ,17 083 744-17053 7147 hei_dopTCTG.
=
22 F . no - graft ' .
23 F AML T _ '1701137544-170537547 net duiTCTql 17029/S47 he1 dupTG1V:
25 M no graft, T + =
I I I
=
i 26 F no %ne . - .t =
, =
27 , M AML -. , =
28 F inultillnevlo,T - ., I 7083714 4470837547 het dupTCTG
, 29 F Ma (nts - ,T , - !I70587544-170857547 hel dug=
, 50 M AM1, no gref; + -, ' 51. M AML. . - + 170337544-1701375475c0upia ' 92 M muldlinealla - - -. 55 F no gra fr, T

=
= . _ , 34 . M no graft,' ¨ . , .
=
35 F , AML, no graft- , 1.7003754 4.170037547 lei_dopTCRI
=
=
, 55; M . ANIL, multillnaage= 170837144470897547 hel_dtipTCTG, =
, 87 F AML 41 7083754 447083754 7 he t_duplCIG;
=
=
. 51 M multIlineage, no graft , .170137144.17012754 7 he i_clupTCTC
=
=
' 99 F . multIlInnage ...
170137144.17081714 7 hei_ctopTCTG
=
=
, 40 . F AML, no ,rtift - =
, 41 , M . AML- . - -42 F MeltIllneage- - 1 701197543_1708375 4 ThainiatCGCG
.
, 43 M no graft - - .
.
44 Ni multIllniage- = - . -, 45 M AML, I1ML +7- - . .170337544-170137547 htt_111,01CP3 46 F T, no graft . += 174337544-170337547 hoLdopTCTO
=
, 47 F no graft . -49 , Ni multIllneage- - . .
, ' . 50 M ' AML

. - -Si , F . multIlinear - -1,170837544-170837547 het_dupTCTG' -02 . F - PruklIngago, 7 170837544-170137547 het_dellICTG, 03 Ni : multIlimagi . - - 1170837544-17083754711d clupTCTC, , =
54 F . multi/Image -479137544170137547 0ei:dulaTC7G
81 , Ni ; mukihnoago - -!170837544.17082734 7 ha derrOCTC' 56 F . no graft - - ,,1 70 13754 4-170837547 hei:chIpTICIG.
57 P . no - graft -170897544.170537547 'lei deETCTG
. -58 , F Tõ no graft -:170097544470837547 hai¨dgfICTG
59 M - no . - graft .
170837544.170837547 het_dupTC1G
AML,multIlInnago . - _ -.. , ,....,, ..... . '170 7544.070037147 het deETCTG
=

62 F AML . =
63 Ni mohllineage +. ,, 1700 97543 17013714 4het insTGCT
64 M muftlIneage +- ', 170 897544:170 82754 7 hot duyITCPG
60 Ni no graft . - -170137544.170137547 hm¨dopTc'TG
66 F no graft +- ;170E1375444 70037347 heE¨dopTCTO
67 Ni multIlineage . + . =
. .
.
.
.
61 M no gra ft - , 170817545 17083754 4het latc-70.0 69 F _Awl. __. ...- , ,,,,....... _,....õ........ , ,, . , , A
' - o ------- A11.1 + 170111714-4.1 70 031 7 f;.er clupxCt.o .i 71 Ni . AmL = - , 170637544.I70007141 hoc¨dupTCY0 1 , 72 M multIllnea ge, no graft . +=
'170827344=170651547 heLdupltr0 73 F . AML, =All/near- - .
'170837544.170897347 hoc duffIrTG
i 74 F no graft + - =
' 76 NI . T, no graft- - 170 0 3754 4-1 70 6 3714 7 her ckipTCTG
, 76 M AML, no graft +. . - 17033754J-170837547 her_clupTC1G
77 , Ni multlineage, no graft +. -170037544-170137547 liet_dupTCTG
70 NI AML . + - ' 79 -i" . - AML - =
BD F no g . . - raft 170637544-170137547 hoLdulliaLl , 61 NI no graft-. _ 52 M no graft . _ IS _ F AML, muitllinea ,ge 170637544-170397547 hoc dupTCBG
DOCSTOR: 293511613 CA 0 2 8 42 635 2 014 ¨ 0 2 ¨11 ___________ _ ______________________________________________ CEBFA
Patient V : 556 CEThrA chr14:3875.1244=337939211mANA
CE5PA5hr19,3379224441/p5120 594_569dupACCCGC ' 13 M . 1 14 F 581 fait 1 is . NA. 5120>Ok134A%,./A 402CM14:134A,AJA 1 16 M 1152_1153hct clupACI 1042_1043141 4iipAC 1 17 F 184_16511cLinsA1CG,1038_1040h6t_dIACG 74_75het,insACCO, 921.930ha &ACC 1 . as ro I
19 , F 1 24 . M = - 1.
25 . M 1 26 ' F 290_299bet.
deIGICCATCOAC, 1046_10471/C9_i8FCT12180,1891vc_delGTCCA1COAC, 536_937her_insC1131 1 27 m . 1 26 = F . - 1 25 F -. 1 , , , 30 , M - - 1 , 31 ' M - - , 1 ..
32 M. 1 . .
35 ., F 355hec110õ 966,990ME 441AC,A
2015bal...142;11711.830hatwklACA 1 .
. .

.--35 M 7 = - 1 97 , F - - 2 38 . M = 1 55 F = - 1 40 , F 427 425Mt_dort,1056_10563eT 40,GAG
317_316het_clutfil; 946_9413her dupGAG 1 .

42 , F 10311>113:3241.>1./R 9711YK1:324LTU9. 1 43 , M - 1 46 : F - i 47 ,. F . - 1 49 : M -1 . -.
SO . M = I
51 , F , = 2 52 , F . 1 . , - .
54 . F 1 55 M 3141141_011K 204het duIC 1 , 57 F ' 1 56 F = I
, = , ,. ... . , , õ... ....
. , --. õ .

51 M , 278C>CA1S5C>GX 565C>CA:265YA7X 165 Ci.C.A:56QC/0C
8554CA25517Y/X 1 , 62 F - 1 .
, 63 M. - 1 84 . M = I ' 65 M 1011_1013 beL441GAC, 1091 V> GA3276.4M 901_90 h4L,MOAC, 9310>CA:327R>ltm, =
, 66 . F - 2 , ' 6.7 1 M. 1 .. , .... .. .,...., . . .
66 = M -. = 1 69 F = 1 70 F . 1 , .
71 M , - 2 , 72 M , - - = 1 75 F . = . 1 74 F - - . 1 7$ nit = = 1 76 M - = t õ.. .,.., .. ..., . , ., .. _ .
77 M . - 1 r 7s NI = = 1 , 79 F . 1 60 F - - t 3,1 m - 1 02 ; m . 228her,c1e1C1 SOS 5O9het deIGC
11 ibe t clalC, 395,3991w dalGC 1 .
DOCSTOR: 29351I\3 CA 02 8 42 635 2 014 ¨ 02 ¨11 DNM73.4, ,Dtql91l3Ackr2:2.506694-, Patient* Sex DDIM-13Acilrz:254635.99-35463599 chr2:25463319- , _ 13 M.
- , _ . .
' =
to fail ...........-16 M . -19 i ' 20 r -21 F _ ,. ' ... -29 , F
25 M _ , 26 F . --' 27 M NI= -23 P. _ 29 F_ . , - .
M -91 M = =
32 . M . - .
=
23 F. - -=
94 M . - = .
- =
95 F . - =
-M . . .
37 F. . - , 39 M- - p5458595T,1C:E160W>WIR
99 F, = =
, r . foil' -_ ... = == " ¨

=
=

49 M , Fail 46 . F _ ' 47 F- = fail ' = 4, .
43 F = =
49 M . - -' M. - , .
;
51 F _ . .
52 F =
, 53 M fail. -.
54 9- . , m- - fail 56 F , =
57 F _ =
. -52 F_ . .
. = .
59 . M . ._ =
, , , F. - -51 m NI .
62 9 i _ 63 m '. _ =
&I. m _ . ¨L. ,.. , .. . -M . .
. 66 F -68 M fail F . 25463541603:7145>SIC - -,, ..., ,....
71 M = .
72 M= - - -73 9.
= .
' 74 F . - .
=
M . Fail. _ 75 , M fail_ t .
' 77 PA = .
-72 . M. - .
, 79 F=
- .
, BO F

. -51 M = =
82 M , fail_ ' , 23 F.
. _ .
DOCSTOR: 293511613 2g , 1D112 chr15: 90631119- 11/191 thr2:269113093-Pauent I , sem DNMI3Athr2:25457219=25457143 !

_ 13 . ....,M 264572420>GA:13132R,RiF1..,,7_, _ __p9113,17,4C.:,C4173P,P/H
14 ,. F 1i4.572-436C1':- 5.3211>RIC ' -15 M . - - =
16 M 25457242Cp0A283217.."4/11 , -- .
17 F . - .
=
-' =
=
, 19 F - . -, , 20 , F - = =
' 21 F = ' 2091131120>3A:13211.>11/11 s!
=
22 . F . 254572420>GA:382R>RX . -, 23 1 F _ - -, 24 ! M - ''. 90631949Co=GA:140P>Riq -25 M- - . , , ., 26 , F - - -' , 27 . M . - -' 28 ,F _ 234572420.0M 32Eri../11-,. -29 F . 90631934 G>GA:140R>10q , ---, ar . . .... .... .. ... _ ._._ . _ .... ... ._...
54 ro - -' 25 F 254572431>CT:682R>It/C .
-96 ! M 254572421as.GA:2823>R/H =
-! 37 F 254572420,-GA38211,F1JH -. -, 38 ,, M - - -; 39 F 25457242G>GC:882R,RIP-, -, i=o r--=
' -, 41 m = -. 42 , F 15457241QQA;331R>11111, =
45 M i -=
-44 M =
=
=
45 ' M 2545724317>a3a2R>Ric- .
=
46 . F =
=
47 F. 2091131131>CM13214.>11./C .
43 ' F-- .
.
15 M tall - 2091131131>C1'.13211.3.11/C
!
50 M 90631934G,GA:14092-lq -S2 F¨ ¨ ' " " . = 90631934G>GA-.1401Pliq --5-4- , F-:90631934QA;14011.311.1q =
55 M -, 254572420,41.4:382E,11/11 . .. .
209113113P.CT:1313>R/C
56 F - .
, 57 i F 254572431>M3921U13./C . .
58 P . 15457243C:=6r.8321DIVC
59 M . 90631934CP=OA1403-,11/11 =
, 60 F 25457243C>=592R,FiC .
-, 61 M - .

, ! 906319340>CA14090,K: = , , 64 m- 906119346>GA:140R>Rig -, GS M- 90631934G>GA:140B>R/Q , -GG F. 9063 I 934G>GA:1401>PIQ -67 M 75457242G>CA1321>Bili 209113113C>C1)132RWC
69 M 254572430CA:511214,12/5 . .
= .
69 , F . 254572420,1308320...,9/P =
G

71 M 552>GA:8iiit>11/14 - - . - = ., ."... .... - ......... , . = - 6 -209113-1.12-E;CFA7.13-2 R.,--"IiiH--7i m -75 F209111113bCT1321t?Ille -,,-.--,- õ - --- - - = ,,.....,.... , -. - -. .. =.õ - ,õ. .,-.¨õ ..... --..- ....õ -=..
74 F 90631934 G>GA:14011>WQ -' 75 M 25457242GP.QA:33211>1UR :
, 76 M .
-, 77 M, 99631934GOA:140PaRiq 2091132620> GA;32R>9%
-, 78 M - i 79 F 254571550;44;19n C:=eir- -90 F 254572420>GA8820>M1- -81 m .

-, 83 F= ! = 209119112q,QA132R>11,51 DOCSTOR: 293511613 Reference List 1. Fialkow, P.J. et al. Clonal development, stem-cell differentiation, and clinical remissions in acute nonlymphocytic leukemia. N Engl J Med 317, 468-73 (1987).
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DOCSTOR: 2935116\3

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Claims (20)

CLAIMS:

1. A method for detecting evidence of pre-cancerous cells in a population of cells from a patient sample, the method comprising:
screening the population of cells for a DNMT3a or IDH2 mutation;
determining positive evidence of pre-cancerous cells if the population comprises cells containing a DNMT3a or IDH2 mutation.

2. The method of claim 1, wherein the patient sample is from a patient that has not been diagnosed with cancer.

3. The method of claim 3, wherein the patient is subclinical for cancer.

4. The method of any one of claims 1-3, further comprising screening the population of cells for a NPM1c mutation and wherein the cells in the population do not comprise a NPM1c mutation.

6. A method of cancer therapy or cancer prevention in a patient, the method comprising providing a treatment to the patient that at least partially eliminates cells containing at least one of a DNMT3a or IDH2 mutation.

6. The method of claim 5, further comprising, prior to treatment, obtaining a sample from the patient, screening the sample for cells containing a DNMT3a or IDH2 mutation, and determining the existence of the DNMT3a or IDH2 mutation in the cells.

7. The method of any one of clams 5 or 6, wherein the patient has previously received chemotherapy and/or radiation therapy.

8. The method of any one of claims 5-7, wherein the cells in the sample do not contain a NPM1c mutation.

9. The method of any one of claims 5-8, wherein the treatment comprises the administration of AG-221.

10. A method of assessing a patient for risk of cancer, the method comprising:
screening cells from a patient sample for mutations in DNMT3a or IDH2; and determining a patient is at risk of cancer if the sample contains cells containing the DNMT3a or IDH2 mutation.

11. The method of claim 101 wherein the risk of cancer is the risk of cancer recurrence following cancer therapy.

12. A method of monitoring cancer therapy in a patient, the method comprising:
screening cells from a patient sample for mutations in DNMT3a or IDH2; and determining an effectiveness or progression of therapy, wherein effectiveness or progression is directly related to a decrease in cells in the sample containing the DNMT3a or IDH2 mutation,

13. The method of any one of the foregoing claims, wherein the patient sample is a blood sample.

14, The method of any one of the foregoing claims, wherein the cells or population of cells comprise at least one of hematopoietic stem cells (HSC), megakaryocytic-erythroid progenitors (MEP), multilymphoid progenitors (MLP), common myeloid progenitors (CMP), granulocyte monocyte progenitors (GMP) and mature lymphoid cells.

16. The method of any one of the foregoing claims, wherein the DNMT3a mutation is the R882H mutation or the R137C mutation.

16. The method of any one of the foregoing claims, wherein the IDH2 mutation is the R14OG mutation.

17. The method of any one of the foregoing claims, wherein the DNMT3a or mutation results in loss in function of DNMT3a or IDH2, respectively.

18. The method of any one of the foregoing, wherein the cancer is leukemia, preferably AML.

19. A composition for use in cancer therapy or cancer prevention, the composition comprising a compound that at least partially eliminates cells containing at least one of a DNMT3a or IDH2 mutation, along with a pharmaceutically acceptable carrier.

20. The composition for use of claim 19, wherein the compound is AG-221.