Differentiation Arrest and Leukemogenesis

1
Differentiation Arrest and Leukemogenesis

• Group Meeting
• Dvir Netanely - June 21st, 2005

2
Normal Blood Cells
3
Normal Blood Cells

• The bone marrow produces stem cells (immature
  cells) that develop into mature blood cells.
• There are 3 types of mature blood cells
• White blood cells (leukocytes) are part of the
  immune system.
• Red blood cells (erythrocytes) carry oxygen from
  the lungs to the body's tissues.
• Platelets (thrombocytes) form blood clots that
  control bleeding.
• Normally, blood cells are produced in an orderly,
  controlled way as the body needs them. This
  process is called hematopoiesis.

4
Hematopoietic Differentiation
5
Hematopoietic Differentiation
6
Hematopoietic Differentiation

• Hematopoietic stem cells in the bone marrow can
  either self-renew or give rise to progenitor
  cells that generate precursors of the myeloid or
  the lymphoid lineage.
• The commitment process is characterized by
  massive cell proliferation in the early phase
  followed by successive restriction to distinct
  cell lineages and to cell differentiation.
• These processes are regulated by trans-acting
  factors which activate or repress genes
• Leukemic mutations interfere with transcription
  factor functions, abrogate cell differentiation,
  and support proliferation. As a consequence, the
  blood is flooded with immature, non-functional
  cell types.

http//www.mdc-berlin.de/englisch/research/researc
h_areas/cancer/leutz.htm
7
Leukemia

• The term leukemia refers to cancers of the white
  blood cells.
• Leukemia is a very heterogeneous disease,
  composed of many subtypes.

8
Leukemia Acute vs. Chronic

• In general, leukemias are classified into acute
  (rapidly developing) and chronic (slowly
  developing) forms.

9
Leukemia ALL vs. AML

• Leukemia is also divided by which type of white
  blood cell is affected
• ALL (Acute Lymphoid Leukemia) vs. AML (Acute
  Myeloid Leukemia).
• AML is more difficult to treat in comparison to
  ALL, overall cure rates for AML remain below 60.

10
Acute Leukemias - Statistics

• Combining childhood and adult cases, there are
  11,000 new cases per year in the U.S.A.
• Overall, acute leukemia strikes 5 out of 100,000
  people each year.
• If untreated, 95 of patients will die within one
  year of diagnosis.
• Acute leukemia is the most common cancer of
  childhood.
• AML is 5 times more common than ALL but ALL
  represents 85 of cases in children.
• Thus, the average ALL patient is 4 years old
  while the average AML patient is 60 years old.

11
AML

• Acute myeloid leukemia (AML) is a cancer of the
  myeloid line of white blood cells.
• The malignant myeloid cells, called myeloblasts,
  fail to mature into the different types of blood
  cells.
• The myeloblasts proliferate rapidly, accumulate
  and overtake the number of healthy blood cells,
  spreading into the bloodstream and other vital
  organs. The lack of healthy blood cells results
  in symptoms such as anemia and abnormal bleeding.

12
Leukemia subtypes
Leukemia
Acute
Chronic
Myeloid
Lymphoid
Myeloid
Lymphoid
AML
(CLL)
(CML)
(ALL)
M0
M1
M2
M3
M4
M5
M6
M7
FAB
13
FAB classification system for AML subtypes

• Acute myelogenous leukemia have been divided into
  8 subtypes, M0 through to M7 under the FAB
  (French-American-British) classification system
  based on the type of cell from which the leukemia
  developed and degree of maturity.
• This is done by examining the appearance of the
  malignant cells under light microscopy or
  cytogenetically by characterization of the
  underlying chromosomal abnormality.
• Each subtype is characterized by a particular
  pattern of chromosomal translocations and have
  varying prognoses and responses to therapy.

14
FAB classification system for AML subtypes

• The eight different subtypes are
• M0 (undifferentiated AML)
• M1 (myeloblastic, immature)
• M2 (myeloblastic, mature)
• M3 (promyelocytic), or acute promyelocytic
  leukemia (APL)
• M4 (myelomonocytic)
• M5 (monocytic)
• M6 (erythroid)
• M7 (megakaryoblastic)

15
FAB Classification
16
FAB Classification
17
AML subtypes
FAB, Translocation and Fusion Proteins
Acute myelogenous leukemias (AMLs) are
genetically heterogeneous and characterized by
chromosomal rearrangements that produce fusion
proteins with aberrant transcriptional regulatory
activities. Myriam Alcalay et. al., 2003
www.med-ed.virginia.edu/. ../wcd/myeloid1.cfm
18
Genetic Abnormalities
19
Genetic Abnormalities
Chromosomal Translocation
20
Major Prognostic AML Sub types
Chromosomal translocations resulting in specific
fusion genes are a hallmark of the leukemias
Z Xiao et. al., Leukemia (2001) 15, 19061913
21
t(1517) PML-RAR
KNOW THE SUBTYPES !

• promyelocytic leukemiaretinoic acid receptor
• This fusion PML-RAR protein is responsible for
  preventing immature myeloid cells from
  differentiating into more mature cells.

Chromosomal translocations resulting in specific
fusion genes are a hallmark of the leukemias
Z Xiao et. al., Leukemia (2001) 15, 19061913
22
t(821) - AML1-ETO

• The AML1 gene encodes the DNA-binding subunit of
  the AML1/CBFb core binding factor transcription
  complex, whereas ETO encodes the mammalian
  homologue of the Drosophila protein Nervy.
• AML1 and ETO are both involved in transcriptional
  regulation of genes in hematopoietic precursor
  cells.
• AML1-ETO fusion protein represses genes whose
  transcription is normally activated by AML/CBFb.

British Journal of Haematology, 1999, 106, 296308
23
t(821) - AML1-ETO
24
Inv16 - CBF-MYH11

• core-binding factor smooth muscle myosin heavy
  chain
• The fusion protein blocks transcription of
  differentiation control genes.

25
The AML1-CBFß Transcription Factor

• In normal cells, heterodimeric AML1-CBFß
  transcription-factor complex binds to the DNA
  sequence TGTGGT in the transcriptional regulatory
  region of AML1-regulated target genes and
  activates transcription through the recruitment
  of coactivators.

26
The AML1-CBFß Transcription Factor

• In AML cells with the t(821) translocation, the
  N-terminal part of AML1 fuses with the C-terminal
  portion of ETO.
• The resultant chimeric protein continues to
  interact with CBFß and to bind to the core
  enhancer sequence however, ETO recruits a
  nuclear corepressor complex and results in the
  dominant repression of AML1-regulated target
  genes.

27
The AML1-CBFß Transcription Factor

• Similarly, the CBFß-MYH11 chimeric protein
  encoded by the inv(16) mutation continues to
  interact with AML1 however, instead of allowing
  AML1 to interact with DNA, this chimeric protein
  recruits AML1 into functionally inactive
  complexes in the cytoplasm.

28
MLL fusion genes

• Mixed-lineage leukemia (MLL) fusion proteins.
• There are more than 40 proteins that have been
  found fused to MLL in leukemia patients, and
  different ones can cause leukemia by different
  mechanisms.
• When the transcription factor MLL functions as it
  should, without a fusion partner, it binds to and
  controls the expression of Hox genes, which in
  turn control cell growth and maturation.

29
Proliferation
Legend (A) In a normal resting cell the
intracellular signaling proteins and genes that
are normally activated by extracellular growth
factors are inactive. (B) When the normal cell is
stimulated by an extracellular growth factor,
these signaling proteins and genes become active
and the cell proliferates. (C) In this cancer
cell, a mutation in a proto-oncogene that encodes
an intracellular signaling protein that is
normally activated by extracellular growth
factors has created an oncogene. The oncogene
encodes an altered form of the signaling protein
that is active even in the absence of growth
factor binding.
30
Cooperating mutations in acute leukemia

• No single mutation is sufficient to cause acute
  leukemia.
• Accumulating experimental and epidemiologic
  evidence suggests a model of cooperation between
  two classes of mutations in acute leukemia
• Mutations that confer a proliferative and/or
  survival benefit to hematopoietic progenitors but
  does not affect differentiation.
• Mutations that impair hematopoietic
  differentiation.
• Acute leukemia, characterized by enhanced
  proliferation and survival of cells and impaired
  differentiation, is the consequence of expression
  of both classes of mutations.

Tallman et. Al., Focus on acute leukemias, Cancer
Cell, 2002
31
Pathogenesis and treatment of acute leukemias

• As indicated by the yellow star, intensive
  cytotoxic chemotherapy remains the mainstay of
  treatment for all acute leukemias.
• Good prognosis leukemias are indicated in blue,
  poor prognosis leukemias are in red, and
  intermediate or unknown are in white.
• There are two classes of cooperating mutations in
  acute leukemia, those that confer proliferation
  and/or survival and those that impair
  hematopoietic differentiation.
• Targeted therapies have been developed or are
  being tested for many of these, such as ATRA.

Tallman et. Al., Focus on acute leukemias, Cancer
Cell, 2002
32
Pediatric Leukemia

• Leukemia is the most common childhood cancer (25
  of all childhood cancers in the US are
  leukemias).
• Approximately 60 of children with leukemia have
  ALL (Acute Lymphoid Leukemia), and about 38 have
  AML (Acute Myeloid Leukemia).

33
Gene expression profiling of pediatric acute
myelogenous leukemia
Blood, 1 December 2004, Vol. 104, No. 12, pp.
3679-3687

• Mary E. Ross, Rami Mahfouz, Mihaela Onciu,
  Hsi-Che Liu, Xiaodong Zhou, Guangchun Song,
  Sheila A. Shurtleff, Stanley Pounds, Cheng Cheng,
  Jing Ma, Raul C. Ribeiro, Jeffrey E. Rubnitz,
  Kevin Girtman, W. Kent Williams, Susana C.
  Raimondi, Der-Cherng Liang, Lee-Yung Shih,
  Ching-Hon Pui, and James R. Downing

34
Pediatric AML subtypes

• The reviewed paper focuses on utilizing gene
  expression technology to identify sub-types of
  Pediatric Acute Myeloid Leukemia (AML).

35
Gene expression profiling of pediatric acute
myelogenous leukemia

• Motivation
• Identification of pediatric AML subtypes based on
  gene expression profiles.
• Seek insights regarding the underlying biological
  process of each subtype.
• Sub type identification will enable the
  development tailored treatment protocols
  customized to a certain genetic lesion which will
  hopefully significantly improve AML patient cure
  rates.

36
Samples (Patients)

• 150 samples used
• 130 pediatric
• 20 adult

Major Prognostic sub types
37
Unsupervised cluster analysis of pediatric AMLs
..relatively tight grouping was observed for the
genetic subgroups AML1-ETO, PML-RAR , and MLL
chimeric fusion genes, and for the morphologic
subgroups FAB-M3, M7, and M4/M5. Unexpectedly,
however, AMLs that expressed the inv16-encoded
CBF -MYH11 failed to cluster indicates
significant heterogeneity within the gene
expression profile of these cases.
38
Expression profiles of pediatric AMLs

• Looking for subtype expression signatures using
  Supervised analysis Trying to find genes that
  discriminate between subtypes.
• Applying SAM with FDR5 yielded only 63
  discriminating genes for CBF-MYH11.

39
Expression profiles of pediatric AMLs

• Hierarchical clustering of the top 50
  discriminating genes for each subtype Some
  clusters are more distinct than others.

40
Similarity plot

• Pair-wise comparisons calculated for 130
  pediatric AML samples using the top 50-ranked
  genes for each subgroup as selected by SAM.

CBFb-MYH11, MLL
Heterogeneity among genes
Observed variation could not be completely
explained by differences in the structure of
chromosomal rearrangements, extent of
differentiation, or presence of specific
secondary mutations
41
Examination of the discriminating genes

• Gene annotation may provide biological insights.
• Discriminating genes may be used as therapeutic
  targets, or as unique class-specific diagnostic
  targets.

42
AML subtype-specific class discriminating genes
Representatives of genes significantly
characterizing one specific subtype
43
Expression signature of core-binding factor AMLs
Selected genes that could discriminate the 2
subtypes of core-binding factor leukemias (CBF
-MYH11 and AML1-ETO ) from all other leukemia
subtypes
44
Building a subtype classifier

• Randomly divided the samples to TRAINING and TEST
  sets.
• TRAINING set was used to train a neural network
  in classifying AML subtypes, based on gene
  expression data for the 250 discriminating genes
  identified by SAM.
• Testing the classifier on the test set, overall
  Prediction accuracy of 93 achieved (100 on
  non-MLL samples).

45
Applying the classifier on Adult samples

• The 20 adult samples were used to test the
  classifier, and yielded overall prediction
  accuracy of 90.
• -gtPediatric and adult samples are similar for
  these subtypes, and therefore the classifier is
  useful for adults as well.

46
Prediction of outcome was not significant

• Identifying a gene expressionbased outcome
  predictor that could provide additional
  prognostic information, either independent of or
  within a genetic subtype, would be a significant
  advance.

47
Gene expression profiles of pediatric acute
leukemia with MLL chimeric fusion genes
130 AML 132 ALL 5 T-ALL w. MLL t
Identification of expression signatures
associated with MLL fusion genes irrespective of
lineage (AML/ALL)
A Unsupervised PCA for the 267 samples
samples cluster according to lineage.
48
Gene expression profiles of pediatric acute
leukemia with MLL chimeric fusion genes
130 AML 132 ALL 5 T-ALL w. MLL
Identification of expression signatures
associated with MLL fusion genes irrespective of
lineage (AML/ALL)
B same PCA, MLL rearrangements are colored in
red C Supervised DAV analysis Separation over
gene space between MLL and non-MLL
49
Gene expression profiles of pediatric acute
leukemia with MLL chimeric fusion genes
130 AML 132 ALL 5 T-ALL w. MLL t
Identification of expression signatures
associated with MLL fusion genes irrespective of
lineage (AML/ALL)
D Top 50 separating genes (MLL vs. non-MLL)
ranked by SAM.
50
Summary I

• Distinct gene expression signatures associated
  with the most common AML translocations were
  identified.
• Gene expressionbased classifier performs with
  93 accuracy in predicting specific
• The classifier performs equally well on AML
  samples obtained from adults.

51
Summary II

• The classifiers inability to correctly classify
  a few samples appears to be a result of molecular
  heterogeneity in AMLs with either CBF -MYH11 or
  MLL translocations. This raises interesting
  questions for further study.
• DAV analysis showed that ALL and AML samples that
  include MLL rearrangements are clustered
  together implying that MLL-rearranged T-ALL and
  AML are biologically more similar to other
  leukemias of similar lineage.

52

• Differentiation Therapy

• Differentiation therapies are broadly defined as
  those that induce malignant reversion (i.e. the
  malignant phenotype becomes benign).
• Clinically, these therapies have been most
  successful for acute promyelocytic leukemia
  (APL), with the use of all-trans retinoic acid
  (ATRA). This treatment has changed a cancer with
  a previously dismal outcome into one of the most
  treatable forms of leukemia.
• The exact mechanisms of differentiation are
  unknown it is unclear if it occurs by inducing
  terminal differentiation (G0 arrest), by inducing
  differentiation backwards to the non-malignant
  form of the cell, or by triggering apoptosis. It
  is likely that it involves all of these pathways

Alexander I Spira et. Al., Differentiation
therapy, Current Opinion in Pharmacology 2003
53
Differentiation Therapy

• Although there are probably mechanistic
  differences in how the various agents lead to
  differentiation, the overall process itself is
  likely to function by allowing malignant tumor
  cells to revert to a more benign form, in which
  their replication rates are lower compared with
  malignant forms, leading to a decreased tumor
  burden
• They might also have a decreased tendency for
  distant metastatic spread, and the process may
  also restore traditional apoptotic pathways, all
  of which could improve a patients prognosis

Alexander I Spira et. Al., Differentiation
therapy, Current Opinion in Pharmacology 2003
54
An Optimistic Ending
The AML subtype M3 (APL) Example
55
AML subtype M3 APL
Acute promyelocytic leukemia (AML M3) is now the
most frequently curable acute leukaemia in adults
if promptly diagnosed and adequately
treated. Parmar S, Tallman MS. , 2003
acute promyelocytic leukemia
M3
t(1517)
PML-RARa
www.med-ed.virginia.edu/. ../wcd/myeloid1.cfm
56
A cure for AML M3 subtype

• All-trans retinoic acid (ATRA) is a drug used for
  the treatment of acute promyelocytic leukemia
  (AML subtype M3).
• It works in AML-M3 because most cases of this
  involve a chromosomal translocation of
  chromosomes 15 and 17, which causes genetic
  fusion of the retinoic acid receptor (RAR) gene
  to the promyelocytic leukemia (PML) gene.
• This fusion PML-RAR protein is responsible for
  preventing immature myeloid cells from
  differentiating into more mature cells.
• This block in differentiation is thought to cause
  leukemia. ATRA acts on PML-RAR to lift this
  block, causing the immature promyelocytes to
  differentiate to normal mature blood cells.

57
ATRA mechanism
Leukemogenic Effects of PML-RARá and Mechanisms
of ATRA/Arsenic Trioxide in the Treatment of APL

•  (A) In the absence of RA, RARa/RXR heterodimers
  recruit the transcription corepressor (CoR),
  which mediates transcriptional silencing by
  mechanisms that include direct inhibition of the
  basal transcription machinery and recruitment of
  chromatin-modifying enzymes. Chromatin
  modification includes histone deacetylation,
  which leads to a compact chromatin structure that
  impairs the access of transcriptional activators.
• In the presence of physiological concentrations
  of RA, the corepressor is released and the
  coactivator is recruited to the RARa/RXR
  heterodimer, resulting in histone acetylation and
  overcoming of the transcription blockage.

• http//medicine.plosjournals.org/perlserv/?request
  slideshowtypefiguredoi10.1371/journal.pmed.00
  20012id20372

58
ATRA mechanism
Leukemogenic Effects of PML-RARá and Mechanisms
of ATRA/Arsenic Trioxide in the Treatment of APL

• (B) PML-RARa fusion protein binds to RARa target
  genes either on its own or with RXR and then
  recruits corepressors, leading to transcriptional
  repression and myeloid differentiation
  inhibition. PML-RARa oncoprotein sequesters the
  normal RXR and PML, inhibits the PML/P53
  apoptotic pathway, and delocalizes PML and other
  proteins from the nuclear body.

• http//medicine.plosjournals.org/perlserv/?request
  slideshowtypefiguredoi10.1371/journal.pmed.00
  20012id20372

59
ATRA mechanism
Leukemogenic Effects of PML-RARá and Mechanisms
of ATRA/Arsenic Trioxide in the Treatment of APL

• (C) In the presence of pharmacological doses of
  ATRA or arsenic trioxide, the PML-RARa fusion is
  degraded in ways that are dependent on caspases
  and proteasomes. The degradation of PML-RAa may
  lead to derepression of transcription suppression
  and restoration of PML nuclear body structure.
  The blockade of other signaling pathways is also
  released, and the anti-apoptotic effect of
  PML-RARa is lost. ATRA also induces cyclic AMP
  (cAMP), which reverses the silencing of RXR,
  induces the expression of RA-induced genes and
  cyclooxygenase 1 (Cox 1), inhibits angiogenesis,
  and downregulates tissue factor. Subsequently,
  ATRA induces terminal cell differentiation, while
  arsenic trioxide induces partial differentiation
  and/or apoptosis of APL cells. The effects of
  ATRA and arsenic trioxide are indicated with red
  and blue arrows, respectively.

• http//medicine.plosjournals.org/perlserv/?request
  slideshowtypefiguredoi10.1371/journal.pmed.00
  20012id20372

60
SUMMARY

• Differentiation arrest is an important component
  in the pathogenesis of many cancers.
• Acute myeloid leukaemia (AML) represents an
  excellent example of a cancer that is
  characterized by a differentiation block.
• Specific haematopoietic transcription factors are
  crucial for differentiation to particular
  lineages during normal differentiation, and are
  controlled by specific patterns of expression and
  protein interactions.
• These same transcription factors are frequently
  disrupted in AML.
• Some mechanisms of disruption involve the effect
  of fusion proteins that are generated by
  chromosomal translocations on haematopoietic
  transcription factors.
• In other cases, in the absence of common
  translocations, the transcription factors
  themselves are mutated.
• Characterizing these transcription-factor
  abnormalities has already affected classification
  schemes based on patient outcome and contributed
  to the improvement of AML patient survival rates.
• These transcription-factor pathways also
  represent important targets for future
  therapeutic intervention.

61
THE END
62
PCA Principle component Analysis - AML
63
Molecular identification of CBF -MYH11 fusion
transcripts in an AML M4Eo patient in the absence
of inv16 or other abnormality by cytogenetic and
FISH analyses - a rare occurrence F Ravandi1, S S
Kadkol2, J Ridgeway1, A Bruno2, C Dodge2 and V
Lindgren2

• The prognostic and therapeutic significance of
  karyotype at diagnosis in patients with AML is
  now fully established. Two of the most common
  recurring cytogenetic abnormalities in AML are
  t(821)(q22q22) and the pericentric inversion of
  chromosome 16, inv(16)(p13q22), or its variant
  t(1616)(p13q22). The chromosome 16
  abnormalities, which are closely associated with
  the FAB subtype M4Eo, result in the creation of a
  fusion gene between the smooth muscle
  myosin-heavy chain gene (MYH11) at 16p13 and the
  core binding factor (CBF ) gene at 16q22. The
  fusion protein product, CBF -MYH11, interacts
  with nuclear corepressors, leading to
  dysregulation of transcription.
• Patients with 'CBF leukemias' including those
  with inv(16)/t(1616) account for up to 20 of
  young adult cases of de novo AML. Such patients
  have a more favorable prognosis, particularly
  when treated with intensive postremission therapy
  including high-dose cytarabine. Therefore,
  accurate identification of these patients at
  diagnosis is of therapeutic significance.
• http//www.nature.com/cgi-taf/DynaPage.taf?file/l
  eu/journal/v17/n9/full/2403056a.html

64
Comparison of Cytogenetic and Molecular Genetic
Detection of t(821) and inv(16) in a Prospective
Series of Adults With De Novo Acute Myeloid
Leukemia A Cancer and Leukemia Group B Study By
Krzysztof Mrózek, Thomas W. Prior, Colin Edwards,
Guido Marcucci, Andrew J. Carroll, Pamela J.
Snyder, Prasad R.K. Koduru, Karl S. Theil, Mark
J. Pettenati, Kellie J. Archer, Michael A.
Caligiuri, James W. Vardiman, Jonathan E. Kolitz,
Richard A. Larson, Clara D. Bloomfield

• ACUTE MYELOID leukemia (AML) is a heterogeneous
  disease with regard to the morphology,
  immunophenotype, and genetic rearrangements
  acquired by leukemic blasts.
• Multiple recurrent chromosome and gene
  rearrangements have been identified in AML, and
  these alterations have been correlated with
  biologic and clinical features of the disease
  resulting in delineation of prognostically
  distinct categories of AML.1-6
• One such category is core-binding factor (CBF)
  AML. Leukemic cells of patients with CBF AML most
  commonly contain either t(821)(q22q22) or
  inv(16)(p13q22), chromosome aberrations that
  result in disruption of genes encoding CBF
  subunits, CBF (also known as AML1) or CBFß,
  respectively.1,6
• Translocation (821) leads to the fusion of the
  AML1 gene, located at 21q22, with the ETO gene at
  8q22 and creation of a chimeric gene AML1/ETO.
• Similarly, a fusion gene CBFß/MYH11 is produced
  by juxtaposition of bands 16q22 (containing CBFß)
  and 16p13 (containing MYH11) as a result of
  inv(16) or, less frequently, t(1616)(p13q22).1,6
  
• http//www.jco.org/cgi/content/full/19/9/2482

65

• 10 September 2001, Volume 20, Number 40, Pages
  5695-5707Molecular mechanisms of leukemogenesis
  mediated by MLL fusion proteins
• Figure 3 The minimal transforming domains of MLL
  fusion partners. MLL fusion proteins are shown
  schematically with gray shading of the fusion
  partners. Boxes with red filling delineate the
  minimal regions of partner proteins necessary and
  sufficient for in vitro myeloid immortalization.
  NTC and TA, conserved amino terminal and
  transactivation domains of ENL Bromo HAT,
  bromodomain and histone acteyltransferase domains
  of CBP OM LZ, octapeptide and leucine zipper
  motifs of AF10

66
The FrenchAmericanBritish (FAB) classification,
described approximately 25 years ago, remains the
foundation on which the morphologic diagnosis of
AML and ALL is based
67
SAM Significance Analysis of Microarray
BACK

• SAM is a method for identifying genes on a
  microarray with statistically significant changes
  in expression.
• Developed in a context of an actual biological
  experiment.
• Assign a score to each gene, uses permutations to
  estimate the percentage of genes identified by
  chance.
• Does not assume normal distribution of the data
• SAM works effectively even with small sample
  size.
• Robust, can be adopted to a broad range of
  experimental situations.

Significance analysis of microarrays applied to
the ionizing radiation response \ Virginia Goss
Tusher,Robert Tibshirani, and Gilbert Chu 2001
68
SAM- procedure overview
Sample genes expression
scale
Define and calculate a statistic, d(i)
Generate permutated samples
Estimate attributes of d(i)s distribution
Identify potentially Significant genes
Choose ?
Estimate FDR