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Subtypes of Acute myeloid leukemia

Subtypes of Acute myeloid leukemia


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I am a computer scientist with no biological background and working on analyzing lab results of patients with Acute myeloid leukemia. They have been tagged with following subtypes of AML:

  • AML with No maturation
  • AML with granulocytic differentiation
  • AML with monocytic differentitaion.

I know there are two classifications for AML subtypes: FAB(M0-M7) and WHO. I think these patients have been classified using FAB, but their corresponding subtypes' names do not match in any of the names associated with M0-M7: http://www.cancer.org/cancer/leukemia-acutemyeloidaml/detailedguide/leukemia-acute-myeloid-myelogenous-classified.

I checked a lot of literature but still not sure about the class of the last two tags in FAB classification. Could someone please help me with that?

  • "AML with No maturation" is M1?
  • "AML with granulocytic differentiation" is M2 (or M4, M5) ?
  • "AML with monocytic differentiation" is M4 or M5 or both ?

Acute myeloid leukemia

Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal cells that build up in the bone marrow and blood and interfere with normal blood cell production. [1] Symptoms may include feeling tired, shortness of breath, easy bruising and bleeding, and increased risk of infection. [1] Occasionally, spread may occur to the brain, skin, or gums. [1] As an acute leukemia, AML progresses rapidly and is typically fatal within weeks or months if left untreated. [1] [6]

Risk factors include smoking, previous chemotherapy or radiation therapy, myelodysplastic syndrome, and exposure to the chemical benzene. [1] The underlying mechanism involves replacement of normal bone marrow with leukemia cells, which results in a drop in red blood cells, platelets, and normal white blood cells. [1] Diagnosis is generally based on bone marrow aspiration and specific blood tests. [3] AML has several subtypes for which treatments and outcomes may vary. [1]

AML typically is initially treated with chemotherapy, with the aim of inducing remission. [1] People may then go on to receive additional chemotherapy, radiation therapy, or a stem cell transplant. [1] [3] The specific genetic mutations present within the cancer cells may guide therapy, as well as determine how long that person is likely to survive. [3]

In 2015, AML affected about one million people and resulted in 147,000 deaths globally. [4] [5] It most commonly occurs in older adults. [2] Males are affected more often than females. [2] The five-year survival rate is about 35% in people under 60 years old and 10% in people over 60 years old. [3] Older people whose health is too poor for intensive chemotherapy have a typical survival of five to ten months. [3] It accounts for roughly 1.8% of cancer deaths in the United States. [2]


Adult AML in Remission

If your doctor tells you that you're in remission, it's a sign that your cancer is under control. For AML, it means:

  • Your CBC is normal
  • Less than 5% of your bone marrow cells are leukemia cells
  • You have no signs of leukemia in your body

Just because you're in remission doesn't mean you won't need treatment. You might enter a phase of treatment that your doctor calls "post-remission therapy." The goal is to get rid of any leukemia cells that may still be in your body.

Continued

Your doctor may want to continue your chemotherapy. They may also suggest radiation therapy at this time. Radiation uses different kinds of energy waves to get rid of cancer cells or stop them from growing.

A stem cell transplant is another treatment option. First, you get chemotherapy and possibly radiation therapy to kill your cancer cells. This process also kills healthy cells, so after that initial treatment you need to get stem cells (immature blood cells) to help create new healthy blood cells. Doctors will either use your own stem cells for the transplant or get stem cells from a donor.


Classifying AML Subtypes

The FAB classification system has been around since the 1970s, but the subtyping process has changed a couple of times in recent years. The WHO classification system became standard in 2008, grouping people based on genetic changes that underlie their cancer (called "driver mutations").

Then, in 2016, pivotal research came out in the New England Journal of Medicine (NEJM) that has taken subtyping even further.  

This study demonstrated that the WHO molecular classifications don't work well for nearly half of AML cases—48% of study participants couldn't be classified based on the WHO molecular groups, even though 96% of them did have driver mutations.

Investigators have now begun reevaluating genomic classification of AML from the beginning, based on:

  • The discovery of many new leukemia genes
  • The discovery of multiple driver mutations per patient
  • Complex mutation patterns

FAB Classification of AML

More than 40 years ago, a group of French, American, and British leukemia experts divided AML into subtypes M0 through M7 based on the type of cell the leukemia develops from and how mature the cells are.

  • M0 through M5 all start in immature forms of white blood cells.
  • M6 starts in very immature forms of red blood cells.
  • M7 starts in immature forms of cells that make platelets.
SUBTYPE SUBTYPE NAME % OF AML DIAGNOSES PROGNOSIS VS. AML AVERAGE
M0 Undifferentiated acute myeloblastic 5% Worse
M1 Acute myeloblastic with minimal maturation 15% Average
M2 Acute myeloblastic with maturation 25% Better
M3 Acute promyelocytic (APL) 10% Best
M4 Acute myelomonocytic 20% Average
M4 eos Acute myelomonocytic with eosinophilia 5% Better
M5 Acute monocytic 10% Average
M6 Acute erythroid 5% Worse
M7 Acute megakaryoblastic 5% Worse
Source: Canaani et al.

WHO Classification of AML

The FAB classification system is still commonly used to group AML into subtypes however, knowledge has advanced with respect to factors that influence prognosis and outlook for various types of AML.

Some of these advances were reflected in the 2008 World Health Organization (WHO) system, which divides AML into several groups:  

  1. AML with myelodysplasia-related changes
  2. AML related to previous chemotherapy or radiation
  3. Myeloid sarcoma (also known as granulocytic sarcoma or chloroma)
  4. Myeloid proliferations related to Down syndrome
  5. AML with chromosomal translocations and inversions
  6. AML not otherwise specified
  7. Undifferentiated and biphenotypic acute leukemias

Groups 5, 6, and 7 are further broken down.

AML With Chromosomal Translocations and Inversions

In chromosomal translocations, a portion of the genetic material breaks off of its original location and re-attaches itself to a different chromosome. In inversions, a segment comes out, flips upside down, and reattaches to its original chromosome.

At least seven types of AML include translocations, inversions, or similar genetic abnormalities.  

AML Not Otherwise Specified

Cases of AML that don't fall into one of the above groups are classified similarly to the FAB system.  

FAB SUBTYPE WHO SUBTYPE NAME
M0 AML with minimal differentiation
M1 AML without maturation
M2 AML with maturation
M4 Acute myelomonocytic leukemia
M5 Acute monocytic leukemia
M6 Acute erythroid leukemia
M7 Acute megakaryoblastic leukemia
-- Acute basophilic leukemia
-- Acute panmyelosis with fibrosis

Undifferentiated and Biphenotypic Acute Leukemias

These are leukemias that have both lymphocytic and myeloid features. They're sometimes called:

  • Acute lymphocytic leukemia (ALL) with myeloid markers
  • AML with lymphoid markers
  • Mixed acute leukemias  

New Classifications: The NEJM Study

The 2016 study that's prompted recent change included 1,540 people with AML. Researchers analyzed 111 genes known to cause leukemia, with the goal of identifying “genetic themes” behind the development of the disease.  

They found that participants could be divided into at least 11 major groups, each with different clusters of genetic changes, and with different disease characteristics and features.

According to the study, most people had a unique combination of genetic changes driving their leukemia, which may help to explain why AML survival rates vary widely. Thus, the researchers worked to develop a new AML classification system using this emerging information.

They concluded that three subgroups exist that weren't accounted for in the WHO classification system. They're called:

Using the proposed system to classify the 1,540 study participants:

  • 1,236 people with driver mutations could each be classified into a single subgroup
  • 56 patients met the criteria for two or more subgroups
  • 166 people with driver mutations remained unclassified

The authors recommended that, in the short term, five specific genetic types (called TP53, SRSF2, ASXL1, DNMT3A, and IDH2) should be incorporated into prognostic guidelines because they're common and strongly influence outcomes.  

Prognostic vs. Diagnostic

The NEJM researchers called for two separate classification systems:

They say the diagnostic system should be based on fixed properties while the prognostic system should change regularly based on available treatments.  

Newer Research

Based largely on the NEJM study, other researchers have investigated certain genetic profiles of AML. According to studies published in 2020, some researchers have identified:

  • Potential new early diagnostic methods for certain subtypes  
  • Potential new ways to identify people likely to be drug-resistant  
  • Potential new combinations of treatments for drug-resistant cases  

One study identified a new drug that researchers say is effective against drug-resistant AML subtypes and, once it's in use, "will have an immediate clinical impact."  


Subtypes of Acute myeloid leukemia - Biology

Presentation during EHA2021: All e-poster presentations will be made available as of Friday, June 11, 2021 (09:00 CEST) and will be accessible for on-demand viewing until August 15, 2021 on the Virtual Congress platform.

Type: E-Poster Presentation

Session title: Acute myeloid leukemia - Biology & Translational Research

Background
The proportion of adult non-M3 AML patients with poor prognosis is relatively large, which is mainly restricted by the current clinical detection limits and sensitivity. To explore new markers that can realize rapid screening and sensitive diagnosis of de novo non-M3 diagnosis of AML subtypes has important clinical significance.

Aims
The aim of this study is to explore serum-based markers in non-M3 AML patients after developing a synthetic approach on Ag nanoparticles-based surface-enhanced Raman spectroscopy (SERS) and non-targeted proton NMR metabolomics.

Methods
According to FAB classification, the serum samples were prepared from five types of non-M3 AML patients (including M0

M2, M4, and M5, n=50 for each type) and healthy controls (n=50, age and gender matched). The Ag substrate nanoparticle-serum complex was first prepared for SERS measurement. The serum metabolites were identified by 1D 1 H NMR method. Statistical methods were utilized in unsupervised and supervised analyses. Both SERS band-intensity data and the metabolite-concentration data were set to merge in classification model for determination of relative diagnostic markers. The receiver operating characteristic (ROC) curves were used to assess the performance of these markers.

Results
We found significant changes in non-M3 AML patients with different subtypes. These appeared in tryptophan, serine, phenylalanine, proline and valine, as well as a variety of nucleic acid bases, phospholipids, D-mannose and collagen. These components were specifically characterized according to vibration mode due to their secondary structure. Several amino acids including tryptophan, serine, phenylalanine, proline and valine, as well as D-mannose were shared in serum metabolites analysis. These metabolites/components were shown to be the key to identify different AML subtypes. Further metabolic pathway analysis suggested disorders in multiple pathways, among which all five types can be separated when SERS and NMR data were set to merge for classification. Typically, the M5 AML was found to be abnormally active in glutathione metabolism, which may show some specificity for diagnosis, but its underlying mechanism still need to be addressed.

Conclusion
We have proposed the combined analysis of serum-based optics and metabolomics to provide rapid screening markers for AML patients with non-M3 diagnosis. It was also demonstrated that this method was of great value in clinical application with acceptable accuracy and good reproducibility.

Keyword(s): Acute myeloid leukemia, Nanoparticle

Presentation during EHA2021: All e-poster presentations will be made available as of Friday, June 11, 2021 (09:00 CEST) and will be accessible for on-demand viewing until August 15, 2021 on the Virtual Congress platform.

Type: E-Poster Presentation

Session title: Acute myeloid leukemia - Biology & Translational Research

Background
The proportion of adult non-M3 AML patients with poor prognosis is relatively large, which is mainly restricted by the current clinical detection limits and sensitivity. To explore new markers that can realize rapid screening and sensitive diagnosis of de novo non-M3 diagnosis of AML subtypes has important clinical significance.

Aims
The aim of this study is to explore serum-based markers in non-M3 AML patients after developing a synthetic approach on Ag nanoparticles-based surface-enhanced Raman spectroscopy (SERS) and non-targeted proton NMR metabolomics.

Methods
According to FAB classification, the serum samples were prepared from five types of non-M3 AML patients (including M0

M2, M4, and M5, n=50 for each type) and healthy controls (n=50, age and gender matched). The Ag substrate nanoparticle-serum complex was first prepared for SERS measurement. The serum metabolites were identified by 1D 1 H NMR method. Statistical methods were utilized in unsupervised and supervised analyses. Both SERS band-intensity data and the metabolite-concentration data were set to merge in classification model for determination of relative diagnostic markers. The receiver operating characteristic (ROC) curves were used to assess the performance of these markers.

Results
We found significant changes in non-M3 AML patients with different subtypes. These appeared in tryptophan, serine, phenylalanine, proline and valine, as well as a variety of nucleic acid bases, phospholipids, D-mannose and collagen. These components were specifically characterized according to vibration mode due to their secondary structure. Several amino acids including tryptophan, serine, phenylalanine, proline and valine, as well as D-mannose were shared in serum metabolites analysis. These metabolites/components were shown to be the key to identify different AML subtypes. Further metabolic pathway analysis suggested disorders in multiple pathways, among which all five types can be separated when SERS and NMR data were set to merge for classification. Typically, the M5 AML was found to be abnormally active in glutathione metabolism, which may show some specificity for diagnosis, but its underlying mechanism still need to be addressed.

Conclusion
We have proposed the combined analysis of serum-based optics and metabolomics to provide rapid screening markers for AML patients with non-M3 diagnosis. It was also demonstrated that this method was of great value in clinical application with acceptable accuracy and good reproducibility.


Treatment Treatment

FDA-Approved Treatments

  • Glasdegib(Brand name: Daurismo) - Manufactured by Pfizer, Inc.
    FDA-approved indication: November 2018, glasdegib (Daurismo) was approved in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are >75 years old or who have comorbidities that preclude use of intensive induction chemotherapy .
  • Enasidenib(Brand name: Idhifa) - Manufactured by Celgene Corporation
    FDA-approved indication: Treatment of adult patients with relapsed or refractory acute myeloid leukemia with an isocitrate dehydrogenase-2 (IDH2) mutation as detected by an FDA-approved test.
    National Library of Medicine Drug Information Portal
    Medline Plus Health Information
  • Gemtuzumab ozogamicin(Brand name: Mylotarg) - Manufactured by Wyeth Pharmaceuticals, Inc., a Pfizer Company
    FDA-approved indication: Mylotarg™ is indicated for the treatment of newly-diagnosed CD33-positive acute myeloid leukemia in adults and treatment of relapsed or refractory CD33-positive acute myeloid leukemia in adults and in pediatric patients 2 years and older.
    National Library of Medicine Drug Information Portal
    Medline Plus Health Information
  • Midostaurin(Brand name: Rydapt) - Manufactured by Novartis Pharmaceuticals Corporation
    FDA-approved indication: Treatment of adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation.
    National Library of Medicine Drug Information Portal
    Medline Plus Health Information
  • Ivosidenib(Brand name: Tibsovo) - Manufactured by Agios Pharmaceuticals, Inc
    FDA-approved indication: July 20, 2018, ivosidenib (Tibsovo) was approved for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible isocitrate dehydrogenase-1 (IDH1) mutation as detected by an FDA-approved test.
    National Library of Medicine Drug Information Portal
  • Venetoclax(Brand name: Venclexta) - Manufactured by AbbVie Inc.
    FDA-approved indication: November 2018, venetoclax (Venclexta) was approved in combination with azacitidine, or decitabine, or low-dose cytarabine for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adults who are age 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy.
    National Library of Medicine Drug Information Portal
    Medline Plus Health Information
  • Cytarabine and daunorubicin liposome injection(Brand name: Vyxeos) - Manufactured by Celator Pharmaceuticals, Inc.
    FDA-approved indication: Treatment of adults with newly-diagnosed therapy-related acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes (AML-MRC)
    National Library of Medicine Drug Information Portal
    Medline Plus Health Information
  • Gilteritinib(Brand name: Xospata) - Manufactured by Astellas Pharma US, Inc.
    FDA-approved indication: November 2018, gilteritinib (Xospata) was approved for the treatment of adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FMS-like tyrosine kinase 3 (FLT3) mutation as detected by an FDA-approved test.
    National Library of Medicine Drug Information Portal

Prognosis of AML

Remission induction rate ranges from 50 to 85%. Long-term disease-free survival is about 20 to 40% overall but is 40 to 50% in younger patients treated with intensive chemotherapy or stem cell transplantation.

Prognostic factors help determine treatment protocol and intensity patients with strongly negative prognostic features are usually given intense forms of therapy followed by allogeneic stem cell transplantation. In these patients, the potential benefits of intense therapy are thought to justify the increased treatment toxicity.

The leukemia cell karyotype is the strongest predictor of clinical outcome. Based on the specific chromosomal rearrangements, three clinical groups have been identified: favorable, intermediate, and poor (see table Prognosis of Acute Myeloid Leukemia Based on Some Common Cytogenetic Abnormalities).

Prognosis of Acute Myeloid Leukemia Based on Some Common Cytogenetic Abnormalities

t(1517)(q24.1q24.1)/PML-RARA

t(1616) or inv(16)(p13.1q22)/CBFB-MYH11

t(821)/(q22q22.1)/RUNX1-RUNX1T1

Karyotype with > 3 abnormalities

Molecular genetic abnormalities are also important in refining prognosis and therapy in AML. Many different mutations exist these are categorized into groups based on their effect on prognosis and treatment. Patients with AML average 5 recurrent gene mutations. Patients with mutations in NPM1, which codes for the protein nucleophosmin, or in CEBPA have a more favorable prognosis. Mutations in FLT3, on the other hand, have a poorer prognosis (including in patients who also have an otherwise favorable NPM1 mutation).

Other factors that suggest a poorer prognosis include a preceding myelodysplastic phase, therapy-related AML, and a high WBC count. Patient-specific adverse prognostic factors include age ≥ 65, poor performance status, and comorbidities. Older patients are more likely to have high-risk cytogenetic abnormalities (see table Prognosis of AML Based on Some Common Cytogenic Abnormalities), secondary AML, and AML that is resistant to multiple drugs.

Minimal residual disease is defined as having < 0.1 to 0.01% (based on the assay used) leukemic cells in bone marrow. In AML, minimal residual disease can be assessed by multiparameter flow cytometry detection of leukemia-associated immunophenotype or by mutation-specific polymerase chain reaction (PCR). These tools are prognostically accurate but are not quite ready for use in clinical practice.


What Is Acute Myeloid Leukemia?

Acute myeloid leukemia has many names, including acute myeloblastic leukemia, acute granulocytic leukemia, acute myelogenous leukemia, and acute nonlymphocytic leukemia. AML is a blood cancer that affects myeloid cells found in the bone marrow, the spongy tissue inside the bones. AML is one of four main types of leukemia, and it is the most common form of acute leukemia in adults.

A fast-growing form of leukemia, AML results in too many immature white blood cells (called myeloblasts) forming in a person’s bone marrow. Healthy myeloid blast cells mature into blood cells: red blood cells, platelets, or white blood cells. In AML, the leukemia cells crowd out the healthy blood cells and can spread to other areas of the body.

AML can occur at any age. However older adults are more likely to develop the disease than younger adults. At least 50 percent of people diagnosed with AML are over 65 years of age. In contrast, AML accounts for around 20 percent of childhood leukemia diagnoses.


Why Does My AML Subtype Matter?

Your AML subtype helps doctors to predict treatment, outcomes, prognosis, and behavior of your disease.

For example, in a study published in 2015, researchers found that types M4, M5, M6, and M7 had the lowest survival rates.   M4 and M5 subtype leukemia cells also are more likely to form masses called granulocytic sarcomas (lesions that form in soft tissue or bone) and to spread to the cerebrospinal fluid (CSF).  

Treatment is the same for most subtypes of acute leukemia with the exception of APL (M3). Different medications are used to treat APL, and the prognosis tends to be better than with other types of acute leukemia.  


Recent advances in acute myeloid leukemia stem cell biology

The existence of cancer stem cells (CSC) was first demonstrated over a decade ago in acute myeloid leukemia (AML) using xenogeneic transplant models.1,2 Since then CSC have been identified in a variety of solid tumors,3 although their existence in some malignancies remains contentious.4,5 AML is a heterogeneous disease, both biologically and clinically, in which a number of distinct genetic abnormalities have been described. However, despite this heterogeneity, early pioneering studies demonstrated that only the most primitive LinCD34CD38 fraction of AML cells and not the more mature LinCD34CD38 or CD34 populations were capable of transferring disease to NOD/SCID mice.2 In the recipient mice, the CD34CD38 cells differentiated into leukemic blasts and recapitulated the phenotype of the disease observed in the patient. Furthermore, these cells were able to reconstitute and give rise to AML in secondary recipients, indicating self-renewal of the LSC in the primary recipients.2 Thus, in a similar way to normal hematopoiesis, it was demonstrated that AML is arranged as a loose hierarchy in which a small population of self-renewing leukemic stem cells (LSC) give rise to a large population of more mature leukemic blasts which lack self-renewal capacity.

This organization helps to explain the all too frequently observed clinical scenario in AML whereby current chemotherapeutic regimens frequently induce remission but relapses, often fatal, are commonly observed. The hierarchical organization of AML suggests that this may relate to current therapeutics targeting only the rapidly proliferating leukemic progenitors, and not the more chemoresistant LSC.6 A thorough understanding of how LSCs differ from their normal counterparts at both the phenotypic and molecular level is, therefore, pivotal to developing targeted therapies, with acceptable toxicities, for AML. This review will aim to describe recent advances in the LSC field with regard primarily to AML, although the concepts described will extend to LSC/CSC in other tumors. The cellular and molecular (both genetic and epigenetic) properties of LSC, and how these might differ from normal hematopoietic stem cells (HSC), will be summarized and potential therapeutic targets explored.

Phenotypic characterization of leukemic stem cells

Initial studies suggested that LSC were restricted to the LinCD34CD38 population, a phenotype shared by the HSC capable of reconstituting normal hematopoiesis in NOD/SCID mice.7 However, since these initial studies, xenograft transplant models have been further refined through the administration of anti-CD122, to suppress Natural Killer cell function, as well as through the use of more immune deficient mouse strains such as NOD/SCID/&beta2m or NOD/SCID/IL2R&gamma mice. Although these refinements did not facilitate engraftment of AML samples which failed to engraft in the original NOD/SCID model,8 engraftment efficiency was increased in the newer models. Furthermore, they also allowed LSC activity to be demonstrated in the more mature LinCD34CD38 progenitor population of some AML patients.9 In addition, intra-femoral, rather than standard tail vein, injection of the transplanted cell innoculum was also found to increase the efficiency of disease transfer.10 Using these refinements LSC were found in more than one compartment and the efficiency of disease transfer followed a hierarchy, with limiting dilution experiments demonstrating that the frequency of LSC was higher in the LinCD34CD38 population compared to the LinCD34CD38 population. However, in both compartments LSC were rare and varied in frequency from 1 in 1.6 ×10 to 1 in 1.1×10 cells.11 These studies suggest that the LSC compartment in AML is more heterogeneous than previously anticipated and includes cells with the surface phenotype of committed progenitors. Importantly, normal LinCD34CD38 progenitor cells are only able to transiently reconstitute NOD/SCID mice and cannot repopulate secondary recipients.12 In fact, studies in ALL may suggest an even greater degree of plasticity within the LSC compartment. In these studies, LSC potential was also demonstrated in populations with phenotypic characteristics of progenitors (CD19 and CD34).13 However, not only were these populations able to transfer leukemia to recipient mice, but CD34 populations were also able to regenerate CD34 progeny within the transplanted leukemia. Similar results within a recently described murine model of AML suggest that AML LSC may also have a comparable developmental plasticity.14

A more detailed comparison of the LSC phenotype with normal myeloid stem and progenitor ontogeny has revealed that in the vast majority of CD34 AML patients, LinCD34CD38CD90CD45RA and LinCD34CD38CD123CD45RA LSC compartments coexist. These respective populations resemble the normal lymphoid-primed multipotent progenitor (LMPP) and granuloctye-monocyte progenitor (GMP) populations at both the phenotypic and molecular level.15 Although the majority of AML cases express the CD34 marker, in some patients, including those with Nucleophosmin (NPM1) mutations, the CD34 percentage is very low. In patients with less than 0.5% CD34 cells, LSC activity was exclusively restricted to the CD34 population, whereas in other patients LSC were present in both the CD34 and CD34populations.16 Together, these studies confirm that the LSC population is phenotypically diverse and can vary markedly between patient subgroups, and even between individual patients within these subgroups. However, how this might reflect the heterogeneity of the initial target cell transformed or the combinations of collaborating mutations is, as yet, unknown.

In addition to CD34 and CD38, LSC have been shown to express a variety of other markers including the myeloid antigens CD33, CD123 and CD13.17 More recently several novel markers that are more highly expressed on CD34CD38 LSC than normal CD34CD38 HSC have been described. These include CLL-1, CD96, TIM3, CD47, CD32 and CD25 (Table 1). C-type lectin-like molecule-1 (CLL-1) was expressed by leukemic blasts at diagnosis from 92% of AML patients analyzed.19 Moreover, although this antigen was expressed on normal CD34CD38 myeloid progenitors, it was absent on normal HSC. However, as with many LSC selective antigens, it is not expressed on every LSC. Within the CD34CD38LSC compartment, a median expression of 33% CLL-1 cells was observed when the data from 29 AML patients were combined.20 CD96 (also known as Tactile) is a member of the Ig gene family. It is also expressed at higher levels in normal progenitors than HSC. Expression was elevated in the CD34CD38 LSC compartment when compared to normal HSC in 65% of AML patients.21 TIM3 is a negative regulator of Th-1-T cell immunity. In addition, the low level of TIM3 expression by HSC compared to LSC enabled the prospective separation of LSC in a variety of AML patients.22 The transmembrane protein CD47 is the ligand for signal regulatory protein &alpha (SIRP&alpha). SIRP&alpha is expressed on phagocytic cells and its interaction with CD47 results in inhibition of phagocytosis. Expression of CD47 by LSC was found to protect them from being phagocytozed by macrophages and dendritic cells and its presence contributed to poor overall survival in patients.23 Although CD47 was consistently more highly expressed by LSC than HSC, there was a large degree of variation across patients in terms of the percentage of LSC that expressed CD47.23 This was also true for the recently identified LSC-specific markers CD25 and CD32, which were found on 34.4% and 24.6%, respectively, of LSC from 61 AML patients analyzed.24 Thus, despite the identification of novel LSC-specific markers, there is a large degree of heterogeneity in expression of these markers among patients. Thus, patient-specific targeting of LSC surface antigens may be necessary.

Table 1. Summary of cell surface marker expression on hematopoietic stem cells and acute myeloid leukemia LSC.

Molecular characterization of leukemic stem cells

LSC share several properties with normal HSC, including a generally quiescent cell cycle status, apoptotic resistance and the capacity to self-renew. Several pathways have been shown to mediate self-renewal in both LSCs and HSCs including WNT/&beta-catenin, NOTCH and Hedgehog signaling, as well as several members of the clustered HOX gene family and the polycomb group protein Bmi1.25,26 However, in AML and other malignancies, these genes or pathways are often mutated, activated or aberrantly expressed. Thus, a therapeutic window may exist, whereby interfering with these pathways might ablate LSC while sparing the normal HSC compartment.

Gene expression profiling studies in mouse and human LSC or pre-leukemia initiating cells have attempted to identify the molecular drivers of LSC function. In particular, mouse models have proven particularly useful in identifying LSC-specific gene expression profiles. Analysis of leukemia models initiated by a range of different MLL-fusion proteins revealed that LSC were enriched within the c-Kit compartment.27 These cells were able to transfer disease to secondary recipients with greater efficiency than c-Kit cells. Comparison of the gene expression profiles of the c-Kit LSC-enriched populations versus the c-Kit- cells, revealed that LSC in these models expressed a gene signature more akin to embryonic stem cells than adult HSC.28 Another group purified LSC to near homogeneity from leukemias induced by expression of MLL-AF9 in the GMP compartment.29 In this model LSC resembled GMP at the phenotypic and molecular level but expressed a set of genes normally restricted to HSC, designated the self-renewal signature. This signature included various Hox genes, including Hoxa9, Hoxa10 and Meis1. These genes are highly expressed in human AML with MLL-translocations and have been shown to regulate the survival and self-renewal of LSC.30,31 This self-renewal signature was partially shared by LSC generated from a completely different initiating mutation: loss of the CEBPA p42 isoform.32 The overlap in gene expression profiles of MLL-AF9 and Cebpa deficient LSC suggests the existence of common mechanisms of progenitor transformation. This idea was further extended to assess gene expression changes in pre-leukemia and leukemia stem cells following expression of a number of disparate AML-associated initiating oncogenes (AML1-ETO, NUP98-HOXA9 and MOZ-TIF2). Despite heterogeneity with regard to the initiating mutation, common and overlapping downstream genes were identified including Bmi1, Meis1, Sox4, Tcf4, Hoxa9 and Smad7.33 Interestingly, some of these genes were able to partially phenocopy the original mutation when over-expressed in murine bone marrow cells. Taken together, these findings suggest that common pathways which facilitate leukemic transformation exist downstream of a variety of different initiating mutations and identifies these pathways as potential therapeutic targets.

Although gene expression analysis of bulk primary human samples has greatly informed the classification and biology of AML, few studies have been performed in human LSC. Nonetheless, a small number of recently performed studies have reported LSC-specific gene expression profiles generated from patient samples.11,15,34&ndash36 All these studies compared gene expression in populations enriched for LSC, as either demonstrated by function or inferred by surface phenotype, with either AML populations which lack LSC properties and/or normal hematopoietic stem and progenitor cells (HSPC) of identical surface phenotype. The studies demonstrate that LSC populations retain similarities of gene expression with the equivalent phenotypic normal HSPC compartment, again reinforcing the possibility that both HSC and progenitors may be the targets of transformation in AML (see below). In addition, they demonstrate that differential gene expression distinguishes LSC populations from those that lack LSC activity. Utilizing the differential LSC signatures, these studies were also able to identify a priori poor risk cases of AML from bulk gene expression profiles, with their predictive value independent of other known prognostic markers, including karyotype and mutational status for FLT3, NPM1 and CEBPA.11,34,35 It is hoped that these signatures may also identify potential drivers of LSC function and putative molecular LSC targets. In support of this concept, in one study, pathways involved in adherens junction, actin cytoskeleton regulation, apoptosis, MAPK and WNT signaling were dysregulated in LSC.36 The identity of these genes contained within the signatures, which include ERG, MEIS1, MECOM (EVI1), HOXA5, MEF2C and SETBP1, is also supportive of their role in leukemogenesis. However, the signatures from the three studies11,34,35 contained, at best, a modest overlap, although FLT3 and HLF were within this overlap. This may reflect molecular heterogeneity within the LSC compartment but likely also reflects the small numbers of profiles assessed and differences in methodology and bioinformatic analysis. It is hoped that an increase in numbers of LSC gene expression profiles and standardization of their analysis will deconvolute these signatures further, allowing critical pathways to be revealed.

The cell of origin in acute myeloid leukemia

Although it is tempting to infer information about the cell of origin in AML based on the cellular phenotype of the LSC, it may be misleading to do so. It is entirely possible that the initial transforming event results in aberrant surface marker expression on this pre-leukemic LSC, such that it is phenotypically uncoupled from its normal counterpart. Despite this, LSC have been isolated which share the cellular and molecular phenotype of HSC and more committed myeloid progenitors,15,29 demonstrating that, at least to some extent, cell surface marker expression on the LSC is suggestive of the cell type initially transformed.

It remains unclear as to whether the initiating mutation responsible for generating the leukemic clone occurs in an HSC, downstream progenitor cell, or both. Murine retro-viral models have demonstrated that certain leukemia associated fusion oncogenes including MLL-ENL, MOZ-TIF2 and MLL-AF9 are able to transform committed progenitors into LSC.29,37,38 However, when under the control of the endogenous MLL promoter, MLL-AF9 was unable to transform GMP, suggesting that gene dosage may play an important role.19 In addition to MOZ-TIF2 and MLL-AF9, NUP98-HOXA9 and AML-ETO were also able to confer self-renewal properties to committed progenitors, although the latter was unable to transform these progenitors in vivo.33 Interestingly, other oncogenes, including BCR-ABL, FLT3-ITD and co-expression of Hoxa9 and Meis1, were not able to alter the self-renewal properties of progenitors.33,38,40 Instead, Hoxa9 and Meis1 or BCR-ABL were oncogenic only when expressed in HSC.38,40 In accordance with this, in chronic myeloid leukemia (CML), the initiating chromosomal translocation t(922) leading to formation of the BCR-ABL fusion gene occurs in an HSC.41 However, transition of the disease to myeloid blast crisis occurs as a result of additional events accumulated in GMP, including activation of &beta-catenin, which confer self-renewal activity to this compartment.42 CML can also progress to lymphoid blast crisis in a minority of patients. Although it was unclear whether the leukemias studied represented lymphoid blast crisis or de novo acute lymphoblastic leukemia (ALL), the initial cell transformed in P210 BCR-ABL1 ALL was demonstrated to be an HSC, in that the chromosomal rearrangement was present in this phenotypic compartment.43 This contrasts with other cases of ALL, including those with P190 BCR-ABL1 and ETV6-RUNX1 rearrangements in which a committed B-cell progenitor was demonstrated to be the likely origin of disease. In addition, in elegant experiments in primary human ALL cells, ETV6-RUNX1 expression conferred self-renewal activity to the B-cell progenitor compartment.43,44 As has been previously mentioned, a similar situation occurs in de novo AML, where many patients demonstrate functionally defined LSC with the surface phenotype of committed myeloid progenitors.9,15 Thus, it seems likely that in AML and ALL both HSC and committed progenitors may serve as the cell of origin (Figure 1). A prerequisite for this argument is that, in the case of progenitor transformation, the initiating mutation must confer self-renewal activity to that compartment in order for the mutation to be propagated.

Figure 1. The cell of origin in AML. During normal myelopoiesis, HSC differentiate into mature blood cells via progenitor populations in a series of lineage restriction steps. They first give rise to multipotent progenitors (MPP) that in turn differentiate into lymphoid-primed multipotent progenitors (LMPP) and common myeloid progenitors (CMP). Granulocyte-monocyte progenitors (GMP) are formed from either LMPP or CMP whereas only CMP give rise to megakaryocyte-erythroid progenitors (MEP). Mutations may accumulate in the long-lived HSC population that has inherent self-renewal capacity, resulting in the generation of a (pre)-LSC. Alternatively mutations may occur in the aforementioned progenitor populations. However, since these cells inherently lack self-renewal activity, the mutation must confer this capacity to the progenitors in order for the mutation to be propagated in a self-renewing (pre)-LSC.

Evolution of the leukemic stem cell compartment

The finding that multiple populations of LSC may exist within a single patient suggests that the LSC population is neither uniform nor static and may evolve from one cellular phenotype to another depending upon the acquisition of additional genetic or epigenetic alterations (Figure 2). Genetic evolution of the LSC compartment has been most convincingly demonstrated in ALL. The ETV6-RUNX1 translocation is a known initiating event that occurs pre-natally in a subset of childhood B-ALL cases.46 Acquisition of this initiating event results in the generation of a pre-leukemic clone that requires subsequent additional alterations for the development of overt leukemia.47 Studies in twins harboring the ETV6-RUNX1 translocation and non-concordant for the development of leukemia, revealed the presence of a CD34CD38CD19 cancer propagating cell in the leukemic twin which was ancestrally related to a pre-leukemic stem cell found to be clonally expanded in the healthy twin.44 Furthermore, elegant studies in which known copy number alterations (CNAs) were examined by FISH at the single cell level in ETV6-RUNX1 cells, revealed considerable complexity in both the structure and hierarchical organization of multiple independent leukemic sub-clones present within individual patients.48 Another study has posited a similar multi-branching model of clonal evolution of BCR-ABL ALL LSC.49

Studies such as these are rare in AML patient samples. However, the continued presence of the AML1-ETO rearrangement in phenotypic HSC from patients in long-term remission has been demonstrated, suggesting the existence of a pre-leukemic stem cell in certain forms of de novo AML.50 In addition, a recent study, presented in abstract form, has extended this work in residual HSC isolated from presentation AML tumor samples that were fully genotyped by next generation sequencing. In small numbers of patients, this study demonstrated the presence of founder mutations in this apparently normal cellular compartment, identifying the residual &lsquonormal&rsquo HSC compartment as a reservoir of pre-leukemic stem cells which lacked the complete mutational spectrum necessary for full transformation.51 In addition, the demonstration that some AML patients harbor specific mutations at diagnosis (such as FLT3-ITD) which are not present at relapse, or vice versa, further suggests the presence of a pre-leukemic stem cell or of clonal evolution in the LSC compartment.52 Recent work, in which the genomes of AML patients at diagnosis and relapse were sequenced, has provided further insight into the clonal evolution of LSC during disease relapse. Additional mutations were acquired in either the dominant clone at diagnosis or a minor sub-clone that presumably enabled the cells to survive the selective pressures of chemotherapy and contribute to relapse.53 This study demonstrates that elimination of not only the founding clone but also sub-clones derived from it are required for long-term remission and highlights the role that chemotherapy may play in contributing to disease relapse.

Figure 2. The heterogeneity of AML. (A) During disease progression the hierarchical organization of the leukemia and phenotype of the predominant LSC population may change. For example, in CML, LSC possess a Lin &minus CD34 + CD38 &minus HSC-like phenotype during the chronic phase. As the disease progresses towards blast crisis, mutations conferring self-renewal activity to the downstream GMP population occur, such that the predominant LSC population at this stage of disease is CD34 + CD38 + CD123 + CD45RA + GMP-like. (B) The genetic repertoire of the LSC is also subject to change during disease progression. Various selection pressures, such as nutrient deprivation, space limitations, anoxia and, most importantly, chemotherapy may select for cells with mutations that enable them to overcome these bottlenecks. Thus various subsets of genetically distinct LSCs may exist within individual patients. Each colored circle represents a cell and a change in color represents the acquisition of an additional mutation. The number of mutations acquired is shown within the cell prior to progression through the bottleneck. Gray cells represent apoptotic cells. Adapted from Greaves.45 (C) In addition to genetic diversity, there is also likely to be epigenetic diversity within the LSC population (as indicated by the cell with the blue nucleus). However, unlike the acquisition of genetic mutations that is an irreversible process, epigenetic modification is dynamic and the LSC may revert to its original epigenetic status after selective pressures have been overcome.

We have provided evidence that both the surface phenotype and mutational genotype of LSC can evolve with time, particularly under selective pressures such as chemotherapy. It has recently been demonstrated in solid organ tumors that altered epigenetic states may also provide sufficient survival/resistance signals for CSC to negotiate these bottlenecks. In the first of these studies, the self-renewal of melanoma cells was demonstrated to be dependent upon dynamic, rather than hierarchical, regulation of the histone H3K4 demethylase JARID1B.54 The second study extended the role of dynamic chromatin regulation in cancer stem cell adaption, demonstrating that the resistance of lung cancer cells bearing the stem cell markers CD24 and CD133 to erlotinib was mediated not by acquired mutation but by global gene expression changes in association with upregulation of signaling via IGF-1R.55 Furthermore, they demonstrated that IGF-1R signaling up-regulated the histone demethylase KDM5A/JARID1A and that this axis altered global H3K4 methylation and H3K14 acetylation patterns. Taking therapeutic advantage of these findings, the authors were able to restore sensitivity in these cells via the addition of histone deacetylase inhibitors or selective inhibitors of the IGF-1 receptor. Thus dynamic epigenetic changes in CSC may mediate self-renewal and survival.

Aberrant epigenetic regulation is also likely to contribute to the heterogeneity of the LSC compartment in AML. Several epigenetic modifiers are mutated in AML either by chromosomal translocation (including MLL, MOZ and JARID1A56) or by point mutation / deletion (including DNMT3A, EZH2, TET2, IDH1 and ASXL1 for a further review see Fathi and Abdel-Wahab57). In addition, it has been demonstrated that DNA methylation patterns can classify AML patient samples and prognosticate outcome,58 and that DNA methylation levels increased from diagnosis to relapse in 83% of AML patients.59 Taken together these findings suggest that in AML altered epigenetic states play a role in mediating resistance of the LSC to chemotherapy.

Figure 3. Targeting the LSC. Knowledge of the initiating genetic lesion and the downstream driver mutations will allow the delivery of specifically tailored targeted therapies. The same is true for identification of LSC-specific surface antigens. For example, MLL-ENL is a known initiating mutation in AML that results in the generation of a pre-LSC. Subsequent as yet poorly characterized (epi)genetic alterations which further drive the leukemic phenotype are thought to be acquired during the evolution of proper LSC. However, despite the acquisition of additional events, LSC continue to be dependent upon MLL-ENL expression. Therefore, interfering with MLL-ENL activity is a potential therapeutic approach. It is likely that targeting the subsequent driver mutations will also abrogate leukemia growth, although the tumors may be less dependent upon these lesions than initiating mutations. In addition, targeting pathways downstream of the initiating lesion, such as Hoxa9, Meis1 and Myb, is also a valid strategy. An alternate, and not mutually exclusive, strategy would be to target aberrant LSC-selective surface markers with antibodies or antibody conjugates. In reality, it is likely that such targeted therapies would need to be combined with each other and current chemotherapies for maximal effect. But it is hoped that efficient targeting of the LSC compartment could result in either loss of self-renewal, apoptosis or differentiation leading to improved outcomes in patients with AML.

Targeting the LSC compartment?

In order to effectively eradicate AML, drugs which selectively target the LSC compartment with minimum toxicity to the normal HSC compartment are required. Antigens which are selectively expressed by leukemic cells represent one such area of therapeutic intervention, and the use of rituximab, which targets the CD20 antigen, has revolutionized the treatment of lymphoproliferative disorders.60 Initial studies in AML have focused on targeting CD33 which is highly expressed by LSCs and their progeny.61 Gemtuzumab ozogamicin (Myelotarg), an antibody against CD33 conjugated to the toxin calicheamicin, is now in clinical trials.62 Although this agent is effective at inducing remission in some patients, they are still prone to relapse, presumably because LSC are resistant to the toxin.63 Furthermore, safety issues have been raised in at least one other trial and prolonged cytopenias have also been described despite effective clearing of the leukemic cells by the treatment.62 This may reflect the expression of CD33 by normal HSC.17

Several antigens are more highly expressed by AML LSC than normal HSC. This raises the possibility of a therapeutic window whereby therapeutic antibodies could be used to selectively target LSC whilst sparing HSC (Table 1). Indeed, promising results were obtained when some of these antigens were targeted in xenografts. As an example, treatment of AML cells with a neutralizing antibody against CD123 prevented their engraftment in NOD/SCID mice and reduced leukemic cell burden in mice with established disease.64 Similar results were obtained following treatment of AML xenografts with antibodies targeting CD4418 and CD47.23 Importantly, in all cases LSC were selectively targeted by the treatment and HSC were significantly less affected.

An alternative strategy is to target the initiating mutation or molecular pathways directly downstream. As direct proof of principle for this concept, several studies have ablated the initiating mutation in murine models of leukemia, leading to their regression. For example, the MLL-ENL fusion protein is sufficient for both the initiation and maintenance of disease in mouse models.65,66 Ablation of MLL-ENL expression in mice with established AML resulted in disease regression, despite the acquisition of additional mutations.66 Thus, despite evolution of the disease, the leukemic cells remained addicted to the initiating oncogenic lesion, indicating that therapies which interfere with MLL-fusion protein activity would be of therapeutic benefit.

MLL-fusion proteins aberrantly regulate gene expression through a number of interactions with multi-protein complexes including members of the super elongation complex (SEC)67,68 and polymerase-associated factor complex (PAFc)69,70 These associations are mediated, at least in part, through interaction with the bromodomain and extra-terminal (BET) chromatin adaptor proteins.71 Displacement of BET family members from chromatin with a small molecule (I-BET151) which disrupts the protein-protein interaction between BET proteins and acetylated lysine residues in histone tails resulted in apoptosis of immortalized cell lines harboring MLL-fusion genes and enhanced the survival of mice with established leukemia.71 Furthermore, I-BET151 treatment of human LSC from patients with MLL-rearranged AML completely ablated their clonogenic capacity in vitro, while normal HSC were relatively unaffected. Thus, indirect inhibition of MLL-fusion protein activity by preventing its association with chromatin is efficacious in pre-clinical studies.

A similar study demonstrated that knock down or inhibition of the BET family member BRD4 had a profound effect on the growth of human AML cells harboring a variety of mutations.72 Furthermore, gene expression changes upon BRD4 inhibition were similar to those described by the same researchers upon Myb knock down in an MLL-AF9 AML mouse model. Ablation of Myb expression inhibited proliferation of leukemic cells in vitro and eradicated disease in vivo with limited effects on normal hematopoietic cells.73 Thus, Myb appears to be an early and key player in oncogene addiction mediated by MLL-fusion proteins. This and other studies have shown that Myb expression may be a more general mediator of self-renewal in a variety of different sub-types of AML. As an example, Myb was also demonstrated to be part of an immediate self-renewal program downstream of other oncogenes including NUP98-HOXA9 and MOZ-TIF2.33 Therefore, there are likely to be common and overlapping pathways mediating self-renewal downstream of a variety of different initiating mutations that may also offer avenues of therapeutic intervention in a wider group of AML patients.

In summary, it is apparent that the vast heterogeneity evident in AML extends to the LSC compartment at both the cellular and molecular level. However, our knowledge base for LSC biology and how this might differ from HSC biology continues to grow. The further identification of new LSC-specific markers represents a novel therapeutic avenue, although it is becoming apparent that no single marker is uniform for LSC between, and even within, individual patients. In addition, mouse models will continue to be invaluable tools for the study of mechanisms of leukemic transformation, LSC biology and therapy. Gene expression profiles of LSC enriched populations have also begun to provide much needed knowledge about the molecular mechanisms mediating self-renewal of human LSCs. However, the limited overlap in expression signatures between pioneering studies requires further scrutiny. Finally, proof-of-principle studies demonstrate that initiating lesions may be targeted, even those which require inhibition of a protein-protein interaction. Therefore, although targeted patient-specific therapies might still be a considerable way off for the majority of patients, therapies which target specific initiating mutations, their downstream pathways and LSC selective surface antigens are now a reality and clinical trials are underway for these as single agents. However, their long-term status in the treatment of AML will no doubt require their combination with standard chemotherapeutics. These studies are eagerly awaited and will hopefully yield exciting results.


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