Course Presentation - College of American Pathologists

Updated 9/10/14
Where the experts are. Where you want to be.™
S1494 CAP/ASH Algorithm
g
for
Initial Work-up for Acute
Leukemia
Daniel A.
A Arber, MD
James W. Vardiman, MD
 2014 College of American Pathologists. Materials are used with the permission of the faculty.
Where the experts are. Where you want to be.™
CAP/ASH GUIDELINES ON
ACUTE LEUKEMIA
James W.
W Vardiman, MD
Daniel A. Arber, MD
 2014 College of American Pathologists. Materials are used with the permission of the faculty.
Disclosure
• None
3
Course Objectives
• Determine proper sample collection at
the time of diagnosis
• Identify key decision points that trigger
appropriate
i t ttestt ordering
d i
• Apply CAP/ASH recommendations for
test selection
4
Agenda
T i
Topic
Ti
Time
Opening/Introductions
1:30-1:50
Key Questions 1 & 2
1:50 2:00
1:50-2:00
Key Question 3
2:00-2:25
Key Question 4
2:25-2:50
2:25
2:50
Key Question 5
2:50-3:00
y Question 6
Key
3:00-3:15
Summary and Closing
3:15-3:30
5
INTRODUCTION
-
Overview of CAP/ASH process and participants
-
Why we need guidelines for the work-up of acute leukemia
-
Review the key questions and recommended guidelines
INTRODUCTION: CAP PATHOLOGY &
LABORATORY QUALITY CENTER
• The CAP Center develops evidence-based
guidelines (EBGs)
g
(
) and consensus statements
related to the practice of pathology and
laboratory medicine.
• These guidelines are used to improve
diagnostic procedures and testing that will
allow
ll
ffor more informed
i f
d patient
ti t managementt
and improved patient outcome.
© 2014 College of American Pathologists. All rights reserved.
7
Guideline Life Cycle
Submit and Select Ideas
Determine Scope and Form Workgroup
k
Maintain
Research and Review Evidence/Draft Recommendations
Publish and Implement
Review and Review
and
Approve
© 2014 College of
American Pathologists. All
rights reserved.
Solicit Comment
Complete C
l t
Recommendations 8
Some CAP EBGs in Progress
•
CAP/ADASP Interpretive Diagnostic Error Reduction Through Targeted
Case Review in Surgical Pathology and Cytopathology (Complete
Recommendations))
•
CAP/NSH Uniform Labeling Requirements for Slides and Blocks in Surgical
Pathology (Review and Approve)
•
Bone Marrow Synoptic Reporting for Hematologic Neoplasms (Complete
Recommendations)
•
CAP/ASCP/AMP Molecular Markers for the Evaluation of Colorectal
Cancer (Research and Review)
•
HPV Testing in Head and Neck Squamous Cell Carcinomas (Research and
Review)
•
CAP/ASCP HER2 Testing Guidelines for Gastric Cancer (Determine Scope
and Form Workgroup)
•
Development of Validation of Quantitative Analysis for Digital Imaging
(Determine Scope and Form Workgroup)
© 2014 College of American
Pathologists. All rights reserved.
9
CAP/ASH QUESTION
The CAP has
Th
h partnered
t
d with
ith the
th
American Society for Hematology (ASH)
to develop guidelines
i
i
to determine
i
the
following overarching question:
For the initial workup of acute leukemia,
leukemia
including AML, ALL, mixed phenotype acute
g
leukemia, what is the recommended testing
for proper diagnosis and prognosis
determination?
© 2014 College of American
Pathologists. All rights reserved.
10
SCOPE
• The guideline addresses issues in these
four areas:
– Morphology
– Flow cytometry
– Cytogenetics
– Molecular genetics
© 2014 College of American
Pathologists. All rights reserved.
11
Guideline Life Cycle
Submit and Select Ideas
Determine Scope and Form
and Form Workgroup
Maintain
Research and Review Evidence/Draft Recommendations
Publish and Implement
Review and Review
and
Approve
© 2014 College of
American Pathologists. All
rights reserved.
Solicit Comment
Complete C
l t
Recommendations 12
Expert & Advisory Panel Members
Expert Panel
Advisory
i
Panel
Daniel Arber, MD, Co-Chair, CAP
James Vardiman, MD, Co-Chair, ASH
Michael Borowitz, MD, PhD, ASH
Melissa Cessna, MD, CAP
Joan Etzell, MD, CAP
Kathryn Foucar, MD, ASH
Robert Hasserjian, MD, ASH
J Douglas Rizzo, MD, ASH
J.
Karl Theil, MD, CAP
Sa Wang, MD, CAP
Frederick R. Appelbaum, MD
Clara Bloomfield, MD
William. L Carroll, MD
Laura Housley, Patient Advocate
Jerry Hussong, MD
Steven H. Kroft, MD, FASCP
Michelle Le Beau, PhD
Martin S. Tallman, MD
© 2014 College of
American Pathologists. All
rights reserved.
13
CAP staff:
Nicole Thomas, MPH, CT(ASCP): Guideline Development Manager
Tony Smith, MLS, ECMS (AIIM) and ,
(
),
,
Carol Colasacco, SCT(ASCP),MLIS, AHIP: Medical librarians
Bryan Rumble, MSc: Primary contracted methodologist Christina Lacchetti, MHSc: Contracted methodologist to assist with data extraction
with data extraction
((All COIs are managed and vetting through CAP) g
g
g
)
© 2014 College of
American Pathologists. All
rights reserved.
14
Guideline Life Cycle
Submit and Select Ideas
Determine Scope and Form Workgroup
k
Maintain
Research and Review Evidence/Draft Recommendations
Publish and Implement
Review and Review
and
Approve
© 2014 College of
American Pathologists. All
rights reserved.
Solicit Comment
Complete C
l t
Recommendations 15
Research and Review of Evidence:
S t
Systematic,
ti unbiased
bi
d review
i
off the
th medical
di l evidence:
id
•
Scholarly articles from the published literature
•
Expert individuals
•
P f i
Professional
l experience/practice
i
/
ti
•
•
Evidence based clinical practice guidelines, Clinical
Practice Guidelines, Systemic Reviews, Meta-analyses, Randomized
controlled trials, Diagnostic studies, Prospective Cohort Studies
Assessment of quality of individual studies is assessed by
a methodologist
© 2014 College of
American Pathologists. All
rights reserved.
16
Guideline Life Cycle
Submit and Select Ideas
Determine Scope and Form Workgroup
k
Maintain
Research and Review Evidence/Draft Recommendations
Publish and Implement
Review and Review
and
Approve
© 2014 College of
American Pathologists. All
rights reserved.
Solicit Comment
Complete C
l t
Recommendations 17
INTRODUCTION: SELECTION OF THE
QUESTION / EVOLUTION OF THE PROJECT
Q: Why do we need guidelines for the work-up of acute leukemia?
A:
1. Evolving
g clinical and scientific data continually
y influences
approaches to diagnosis, classification, identification of prognostic
factors and therapy in acute leukemia
2. There should be periodic assessments in order to improve
diagnostic procedures and testing that will improve treatment
decisions
© 2014 College of American
Pathologists. All rights reserved.
18
In the `70s & 80s, the diagnosis and classification
of AL was fairly simple; the FAB proposal was the
“guideline” …..
© 2014 College of
American Pathologists. All
rights reserved.
19
`70s and `80s Diagnosis and classification of
AL: FAB
W-G
Acute lymphoblastic
y p
leukemia
L1, L2, L3
MPO
NSE
© 2014 College of
American Pathologists. All
rights reserved.
Acute myeloid leukemia
(M1) AML without maturation
(M2) AML with maturation
(M3) Acute promyelocytic leukemia
(M4) Acute myelomonocytic leukemia
(M5) Acute monocytic leukemia
(M6) Acute erythroleukemia
20
Diagnosis and classification of AL:
FAB with immune markers
W
G
W-G
Acute lymphoblastic leukemia
Acute
lymphoblastic leukemia
L1, L2, L3
MPO
NSE
© 2014 College of
American Pathologists. All
rights reserved.
CD61
Acute myeloid leukemia
Acute
myeloid leukemia
(M0) AML with minimal differentiation
(M1) AML without maturation
(M2) AML with maturation
(M2) AML with maturation
(M3) Acute promyelocytic leukemia
(M4) Acute myelomonocytic leukemia
((M5) Acute monocytic leukemia
)
y
(M6) Acute erythroleukemia
(M7) Acute megakaryocytic leukemia
21
Survival according to FAB guidelines
© 2014 College of American
Pathologists. All rights reserved.
22
Diagnosis and classification of AL:
FAB
W‐G
Acute lymphoblastic leukemia
Acute
lymphoblastic leukemia
L1, L2 Acute myeloid leukemia
(M0) AML with minimal differentiation
(M0) AML with minimal differentiation
(M1) AML without maturation
(M2) AML with maturation
(M3) Acute promyelocytic leukemia
(M3) Acute promyelocytic leukemia
(M4) Acute myelomonocytic leukemia
(M5) Acute monocytic leukemia
((M6) Acute erythroleukemia
)
y
(M7) Acute megakaryocytic leukemia
MPO
NSE
© 2014 College of American Pathologists. All rights reserved. CD61
23
Cytogenetics in AML: Classification
and guidelines
© 2014 College of
MLL break apart probe
American Pathologists. All
rights reserved.
t(8;21)(q22;q22) RUNX1/RUNX1T1
24
NORMAL
t(8;21)
inv(16)
t(15;17)
-7
-5
Complex
Grimwade, et al. Blood 1998;92:2322
The importance of diagnostic cytogenetics on outcome in AML: Analysis
of 1612 patients entered in the MRC AML 10 Trial.
WHO: 2001
1) Utilizes all available information –
clinical findings, morphology,
immunophenotype,
p
yp and genetic
g
features – in an attempt to define
disease entities of clinical
significance
i ifi
2) It is a “consensus” classification
in which experts in the various
hematologic neoplasms met,
debated and the majority agreed
debated,
to the definition and classification
of specific
o
spec c d
disease
sease e
entities
t t es
© 2014 College of
American Pathologists. All
rights reserved.
26
WHO: 2001 Classification Guidelines
Acute Myeloid Leukemia
AML with recurrent cytogenetic abnormalities
t(8;21)
t(15;17)
inv(16)
11q23
AML with multilineage dysplasia
AML/MDS, Therapy‐related
AML, NOS
Lymphoblastic Leukemias
Precursor B‐, T‐ lymphoblastic leukemia/lymphoma
Acute leukemia of Ambiguous Lineage
© 2014 College of
American Pathologists. All
rights reserved.
27
Cooperation Between Mutations in
AML Pathogenesis
Class I Translocations/ Mutations
Class II Translocations/Mutations
FLT3-ITD
FLT3FLT3--TKD
FLT3
KIT
RAS
PTPN11
JAK2
proliferation and/or
survival advantage; not
affecting differentiation
© 2014 Gilliland
College of American
and Griffin,
Pathologists. All rights reserved.
PML
PML--RARA
RUNX1-RUNX1T1
RUNX1CBFB
CBFB--MYH11
MLL fusions
CEBPA
NPM1?
NPM1
?
AML
i
i d hematopoietic
h
t
i ti
impaired
differentiation and
subsequent apoptosis
Blood 100:1532,
2002 (modified by H. Dohner)
28
WHO 2008: Mutations in the
classification of AL
© 2014 College of American
Pathologists. All rights reserved.
29
WHO 2008: Classification and
guidelines
AML with recurrent genetic abnormalities
t(8;21)(q22;q22); RUNX1‐RUNXT1
t(15;17)(q22;q12); PML‐RARA
inv(16)(p13.1q22)/t(16;16)(p13.1q22); CBFB‐MYH11
t(9;11)(p22;q23); MLLT3‐MLL
t(6;9)(p23;q34);DEK‐NUP214
inv(3)(q21q26.2)/t(3;3)(q21;q26.2);RPN1‐EV11
t(1;22)(p13;q13);RBM15‐MKL1
AML with mutated NPM1
AML with mutated CEBPA
AML with multilineage dysplasia
AML/MDS, Therapy‐related
/
© 2014 College of
American Pathologists. All
rights reserved.
AML, NOS
30
WHO 2008: Classification and
guidelines
AML with recurrent genetic abnormalities
t(8;21)(q22;q22); RUNX1‐RUNXT1
t(15;17)(q22;q12); PML‐RARA
inv(16)(p13.1q22)/t(16;16)(p13.1q22); CBFB‐MYH11
t(9;11)(p22;q23); MLLT3‐MLL
t(6;9)(p23;q34);DEK‐NUP214
inv(3)(q21q26.2)/t(3;3)(q21;q26.2);RPN1‐EV11
t(1;22)(p13;q13);RBM15‐MKL1
AML with mutated NPM1
AML with mutated CEBPA
AML with multilineage dysplasia
AML/MDS, Therapy‐related
/
© 2014 College of
American Pathologists. All
rights reserved.
AML, NOS
31
Ley TJ, et al. NEJM 2013
Guideline Life Cycle
Submit and Select Ideas
Determine Scope and Form Workgroup
k
Maintain
Research and Review Evidence/Draft Recommendations
Publish and Implement
Review and Review
and
Approve
© 2014 College of
American Pathologists. All
rights reserved.
Solicit Comment
Complete C
l t
Recommendations 35
CAP/ASH QUESTION
The CAP has
Th
h partnered
t
d with
ith the
th
American Society for Hematology (ASH)
to develop guidelines
i
i
to determine
i
the
following overarching question:
For the initial workup of acute leukemia,
leukemia
including AML, ALL, mixed phenotype acute
g
leukemia, what is the recommended testing
for proper diagnosis and prognosis
determination?
CAP Guidelines for workup of acute
leukemia is built around 6 questions:
Key Question 1: What clinical and laboratory information should
be available during the initial diagnostic evaluation of a patient
with acute leukemia?
Key Question 2: What specimens and sample types should be
evaluated during the initial workup of a patient with acute
leukemia?
Key Question 3: At the time of diagnosis, what tests are
required for all patients for the initial evaluation of an acute
leukemia?
y Question 4: Which tests should be p
performed only
y on a
Key
subset of patients, including in response to results of initial
tests and morphology?
© 2014 College of
American Pathologists. All
rights reserved.
37
CAP GUIDELINES FOR WORKUP OF ACUTE
LEUKEMIA IS BUILT AROUND 6 QUESTIONS:
Key Q
K
Question
ti
4:
4 Which
Whi h tests
t t should
h ld be
b performed
f
d only
l on a
subset of patients, including in response to results of initial tests
and morphology?
Key Question 5. Where should testing be performed?
Key Question 6: How should test results and the
diagnosis be correlated and reported?
© 2014 College of American
Pathologists. All rights reserved.
38
KEY QUESTION 1: WHAT CLINICAL AND LABORATORY
INFORMATION SHOULD BE AVAILABLE DURING THE INITIAL
DIAGNOSTIC EVALUATION OF A PATIENT WITH ACUTE LEUKEMIA?
Essential Information
i. Results of a recent or concurrent complete blood count (CBC) with
leukocyte differential and peripheral smear preparation
ii. Relevant clinical information should include, but not be limited to:
family history
history, age
age, gender
gender, history of prior malignancy),
malignancy) history of
predisposing conditions (Down syndrome, bone marrow failure
syndromes, chronic hematologic disorders) or possible confounding
factors (history of growth factor, B12 and/or folate deficiency) , and
predisposing therapies
iii. Key physical examination findings, including, but not limited to: CNS
status, presence of tumor masses, organomegaly, other tissue lesions
such as cutaneous disease.
Recommended Information
i. Coagulation studies
Useful Information
i. Ethnicity
h i i
ii. Other clinical factors recognized as having prognostic importance,
such as Performance status
iii Results of recent chemistry assays
iii.
© 2014 College of American
Pathologists. All rights reserved.
39
KEY QU
QUESTION
S O #1
Why is the history important to the pathologist?
1. Therapy related MDS/AML/ALL
2. A new subgroup, Familial MDS/AML and related
disorders, is proposed for the WHO 2015 revision
© 2014 College of American
Pathologists. All rights reserved.
40
Therapy‐related Myeloid Neoplasms
‐ IIncludes therapy‐related AML (t‐AML), ALL (t‐ALL), MDS (t‐MDS) and l d th
l t d AML (t AML) ALL (t ALL) MDS (t MDS) d
MDS/MPN (t‐MDS/MPN) that occur as a late complication of cytotoxic chemotherapy and/or radiation therapy administered for a prior neoplastic or non neoplastic disorder
prior neoplastic or non‐neoplastic disorder. ‐ Cytotoxic agents implicated include alkylating agents, RT affecting large fields of active marrow topo II‐inhibitors, anti‐metabolites, anti‐
large fields of active marrow, topo
II‐inhibitors anti‐metabolites anti‐
tubulins WHO 2001
‐ Identification
Identification of cases of t
of cases of t‐MN
MN could be informative for identification could be informative for identification
of cellular pathways affected by known cytotoxic agents and thus contribute knowledge regarding pathogenesis of de novo disease with similar genetic abnormalities.
g
‐ The study of t‐MN may uncover heritable predisposition factors or specific abnormalities in DNA‐repair mechanisms that predispose to t‐MN and could be used for screening/counseling patients prior to d
ld b
df
i /
li
i
i
exposure to similar agents 1. Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Bl d 2002 200 2292 2302
Blood 2002;200:2292‐2302
2. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009;2114:937‐951
© 2014 College of American
Pathologists. All rights reserved.
41
‐ Although t‐MN may be further subclassified into t‐MDS or t‐AML according to the blast count, ‐ Recommended that cases of t‐MDS not be further classified according to criteria for subclassification of MDS de novo
‐ Cases of t‐MN with recurrent genetic abnormalities found in AML de novo should be classified as t‐AML with the recurrent abnormality, e.g., t‐AML with t(9;11)(p21;q23)
WHO 2008
KEY QU
QUESTION
S O #1
Why is history important to the pathologist?
1. Therapy‐related MDS/AML/ALL
2. A new subgroup, Familial MDS/AML and 2
A new subgroup Familial MDS/AML and
Related Disorders, is proposed for the WHO 2015 revision
© 2014 College of American
Pathologists. All rights reserved.
43
Familial MDS/AML Predisposition Syndromes
Familial MDS/AML Predisposition Syndromes
‐ Related to germline mutations in key genes involved in hematopoiesis; usually l d
li
i
i k
i
l di h
i i
ll
demonstrate an autosomal dominant pattern of inheritance
1. “Syndromic associated inherited bone marrow failure syndromes”
1
“S d
i
i t d i h it d b
f il
d
”
Usually occur in pediatric patients with a variety of congenital physical abnormalities, i.e, Dyskeratosis congenita, Fanconi Anemia, Schwachman Diamond syndrome etc
Schwachman‐Diamond syndrome, etc.
2. “Familial MDS/AML Syndromes
Familial predisposition syndromes in which features of
Familial predisposition syndromes in which features of MDS/AML/ALL is often the presenting feature, usually in adults
© 2014 College of American
Pathologists. All rights reserved.
44
Syndrome
Gene
Inheritance
Neoplasm
Other findings
Onset
Familial platelet
disorder with
propensity to
myeloid neoplasms
RUNX1
AD
MDS, AML,
T-cell ALL
Thrombocytopenia,
bleeding due to platelet
dysfunction
Adult
or
Child
Familial AML with
mutated CEBPA
CEBPA
AD
AML
Eosinophilia
Adult
or
Child
Familial AML with
mutated GATA2
GATA2
AD
MDS, AML
Emberger syndrome:
primary lymphedema,
immunodeficiency,
warts
MonoMac syndrome:
pulmonary alveolar
proteinosis,
monocytopenia, NK
and
dB
B-cell
ll decrease,
d
disseminated atypical
myobacterial, viral or
fungal infections
Adult
or
Child
Telomere biology
diseases due to
mutated TERC or
TERT
TERT
TERC
AD
MDS, AML
Macrocytosis,
cytopenia, aplastic
anemia, idiopathic
pulmonary fibrosis,
h
hepatic
ti cirrhosis
i h i
Adult
or
Child
© 2014 College of American
Pathologists. All rights reserved.
45
Case
This 62-year old woman was referred for evaluation of leukopenia and
thrombocytopenia. She had been told these were due to autoimmune
disease but she had no other autoimmune manifestations.
disease,
manifestations During the
initial visit, the patient told the hematologist that her grand-daughter
was ill in another hospital, and was having a bone marrow procedure.
A bone marrow biopsy and aspiration were performed on the patient.
At the time of the bone marrow procedure, WBC=3.9K/uL, Hb=11.5g/dL,
MCV=89fL, Platelets=49K/uL.
Case
Marrow blasts = 6% on differential count
Grand-daughter, AML with monosomy 7 and RUNX1 mutation
Familial Platelet Disorder with a propensity to develop
myeloid malignancy (FPD/AML)
-Autosomal dominant
-Characterized
Characterized by thrombocytopenia
thrombocytopenia, abnormal platelet function
function,
and propensity to develop MDS/AML
-Germline heterozygous mutation of RUNX1
-Mutations are variable, but in FPD/AML most occur in the runt
homology domain (RHD)
Familial Myeloid Disorders
1. Familial platelet disorder with propensity to AML (FPD/AML):
RUNX 1 mutations
2. Familial AML with mutated CEBPA
3. Familial MDS/AML with mutated GATA2
4. ANKRD26‐related thrombocytopenia and myeloid malignancies
Key
yQ
Question 2: What specimens
p
and sample
p types
yp
should be evaluated during the initial workup of a patient
with acute leukemia?
A. Essential in all patients:
i. Fresh bone marrow aspirate 1 Portion used for adequately prepared bone marrow aspirate
1. Portion used for adequately prepared bone marrow aspirate smears
(If an aspirate is unavailable, marrow touch preps of a core biopsy can be used, and an additional core used for ancillary
biopsy can be used, and an additional core used for ancillary studies. If sufficient blasts are present in blood, and if aspirate is unavailable, the peripheral blood may be used for diagnosis and ancillary studies)
y
)
2. Cryopreserved cells or nucleic acid if all ancillary studies cannot be performed on fresh material or if additional studies are required after completion of other studies
ii. Peripheral blood smear with CBC data
© 2014 College of American
Pathologists. All rights reserved.
Key
ey Quest
Question
o 2: What
at spec
specimens
e sa
and
d sa
sample
p e types s
should
ou d be
evaluated during the initial workup of a patient with acute
leukemia
B. Essential in some patients
i. CSF for well‐prepared cytocentrifuge slides and cell count in pts with CNS symptoms or signs
ii. Fresh tissue biopsy in the presence of extramedullary disease in patients with insufficient blasts in marrow or blood
1. Cryopreservation of tissue for future ancillary study
C. Recommended i. Adequate bone marrow trephine bone core biopsy
ii. Bone marrow touch preparations
D. May be useful
i. Marrow clot sections
© 2014 College of American
Pathologists. All rights reserved.
54
Key Question 2: What specimens and sample types should be
evaluated during the initial workup of a patient with acute
leukemia?
Morphologic diagnosis and classification
C t h i t IImmunohistochemistry
Cytochemistry,
hi t h i t
Initial Specimen;
Neoplastic cells from
this specimen are
key to proper
diagnosis, risk
stratification, and
determination of
prognostic factors
Flow cytometry
Aids in diagnosis,
diagnosis classification
Identifies potential therapeutic targets
Establishes a phenotypic fingerprint for
monitoring minimal residual disease
Cytogenetics
Aids in diagnosis, classification
Important for prognosis
May identify therapeutic targets
Provides information regarding
g
g residual disease
Molecular genetics
Aids in diagnosis,
g
classification
May identify potential therapeutic targets
Important for prognosis
Provides information regarding residual disease
11 year old girl with WBC=12.6 K/uL with 20% blasts in the blood. A bone marrow biopsy and aspirate showed >90% blasts. Cytochemistry: Blasts are myeloperoxidase and esterase (ANB) negative
Flow cytometry: CD19+, CD22+, cyCD79A+, HLADR+,
cyIgM +/‐, TdT+/‐, CD33+/‐, CD24+/‐,
CD10‐, CD20‐, CD34‐, CD117‐, CD1a‐, s/cyCD3‐, CD4‐, CD8‐, CD5‐, CD7‐, CD13‐, CD14‐, CD11b‐, CD15‐, CD16‐, CD41‐, Glycophorin A‐, MPO‐
Karyotype: 46, XX [15%]
45, XX, del(11)(q13q24), t(11;19)(q23;p13.3); dic (12;17)(p11.2;p11.2) [35%]
44, idem, ‐13 [50]
Diagnosis: B lymphoblastic leukemia/lymphoma with MLL
l
h bl
l k
/l
h
h
rearrangement © 2014 College of American
Pathologists. All rights reserved.
56
Post induction bone marrow, day 29:
, y
Is there MRD ? P. Blood: WBC=3.2K/uL with no blasts
Bone marrow: 60% cellular with granulocyte, megakaryocytic hyperplasia; 6% blasts with a shift towards immaturity hf
d
in the granulocytic lineage. 6% lymphoid cells with h
hematogones
t
Karyotype: 46, XX [100] FISH N MLL rearrangement/deletion detected
FISH: No MLL
t/d l ti d t t d
© 2014 College of American
Pathologists. All rights reserved.
57
Detection limits (Fraction of leukemic cells): (
)
Microscopy/cytogenetics/FISH: 10‐1
Flow cytometry, Gene expression PCR (e.g., WT1): 10‐4
PCR (translocations, clone‐specific IgH rearrangement, mutations), NGS: 10‐6
Hourigan C, Karp JE Nature Review Clinical Oncology 2013;10:460
© 2014 College of American
Pathologists. All rights reserved.
58
EFS of all patients enrolled on 9900 series therapeutic studies. The 5-year EFS values
plus or minus SE are shown for patients with varying levels of MRD as determined by
f
four-color
l flow
fl
cytometry.
t
t
B
Borowitz
it M J ett al.
l Bl
Blood
d 2008;111:5477-5485
2008 111 5477 5485
©2008 by American Society of Hematology
Two MRD tubes added to regular immunophenotyping panel at diagnosis: © 2014 College of American
Pathologists. All rights reserved.
Day 29 End‐induction bone marrow: Is MRD present?
© 2014 College of American
Pathologists. All rights reserved.
Key Question 2: What specimens and sample types should be
evaluated during the initial workup of a patient with acute
leukemia?
Morphologic diagnosis and classification
C t h i t IImmunohistochemistry
Cytochemistry,
hi t h i t
Initial Specimen;
Neoplastic cells from
this specimen are
key to proper
diagnosis, risk
stratification, and
determination of
prognostic factors
Flow cytometry
Aids in diagnosis,
diagnosis classification
Identifies potential therapeutic targets
Establishes a phenotypic fingerprint for
monitoring minimal residual disease
Cytogenetics
Aids in diagnosis, classification
Important for prognosis
May identify therapeutic targets
Provides information regarding
g
g residual disease
Molecular genetics
Aids in diagnosis,
g
classification
May identify potential therapeutic targets
Important for prognosis
Provides information regarding residual disease
Key Question 3
At the time of diagnosis, what tests are
required
q
for all p
patients for the initial
evaluation of an acute leukemia?
Al
Algorithmic
ith i Approach
A
h to
t AL
Morphologic Review
>20% Blood or
Marrow Blasts
Blast Cell Features
<20% Blood or
Marrow Blasts
Non-Blast Cell Features
Morphology in AL
• Peripheral blood
and bone marrow
differential cell
count
• Blast cell features
• Non-blast cell
features
66
Morphology in AL
• Peripheral blood and bone marrow
differential cell count
– 200 cell count of blood and 500 cell count
of marrow recommended
– Percentage of erythroid cells is also critical
to call erythroleukemia
• Blast cell features
• Non-blast cell features
67
Morphology in AL
• Peripheral blood and bone marrow
differential cell count
• Blast cell features
– Some
S
ffeatures
t
off highly
hi hl suggestive
ti off
specific disease entities
– Other features suggest AML, NOS subgroups
• Non
Non-blast
blast cell features
68
Blast Cell Features
Acute promyelocytic leukemia with t(15;17)(q24.1;q12); PML‐RARA
t(15 17)( 24 1 12) PML RARA
AML with t(8;21)(q22;q22); RUNX1‐RUNXT1
Blast Cell Features
Morphology in AL
• P
Peripheral
i h
l blood
bl d and
db
bone marrow
differential cell count
• Blast cell features
Non-blast
blast cell features
• Non
– Presence or absence of multilineage
dysplasia
– Increases of eosinophils, including abnormal
eosinophils
– Increases in basophils or mast cells
71
AML with Myelodysplasia
Myelodysplasia-Related
Related Changes
• D
Detection
t ti
off multilineage
ltili
dysplasia
– Two non-blast cell lines must
show dysplasia in at least 50% of
cells
o MDS
MDS-related
related cytogenetic
abnormalities or prior MDS
o Absence of the specific genetic
abnormalities
b
liti off AML with
ith
recurrent genetic abnormalities
o Absence of prior history of
therapy
Non Blast Cell Features
Non-Blast
ALL with t(5;14)q31;q32;
IL3‐IGH
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB‐
( ; )(p
;q );
MYH11
AML with t(6:9)(p23;q34);
DEK‐NUP214
Al
Algorithmic
ith i Approach
A
h to
t AL
Morphologic Review
Immunophenotype
Mixed?
Myeloid
Precursor B
Precursor T
Immunophenotyping in AL
• IImmunophenotyping
h
t i
is now expected on
allll acute
t lleukemia
k
i
– Flow cytometry is the
preferred method
– Immunohistochemistry
may be necessary
when the marrow is a
y tap”
p due to
“dry
fibrosis or procedural
problems
Immunophenotyping in AL
• Immunophenotyping
is now expected
p
on
all acute leukemia
• Because of lineage
infidelity, the panel
mustt have
h
multiple
lti l
markers for B, T and
myeloid lineage
Bene MC, Nebe T, Bettelheim P et al. Leukemia 25:567, 2014
Craig FE, Foon KA. Blood 111:3941, 2008
Stetler‐Stevenson M, Davis M, Wood B et al. Cytometry B 72 (Suppl 1):S3, 2007
Immunophenotyping in AL
• Immunophenotyping
–a
allows
o s for
o lineage
eage
assignment in the vast
j y of cases
majority
– Some
immunophenotypes
are highly suggestive
of specific AL
subtypes
Immunophenotyping in AL
• Immunophenotyping
may
y supplement
pp
a
blast cell count, but
should not replace a
manual differential
count when
adequate smears
are available
Early T-Precursor Acute Lymphoblastic
Leukemia (ETP-ALL)
• EEarly T‐Precursor (ETP) ALL comprises 10‐15% of T‐ALL l TP
(ETP) ALL
i
10 15% f T ALL
• Defined immunophenotypically by expression of cCD3, CD7, low CD5 but no CD1a CD4 or CD8
CD5, but no CD1a, CD4 or CD8 • Expresses CD34 and myeloid‐related antigens (CD117, CD33, or CD13) but not MPO • Thought to arise from an early progenitor cell with lineage plasticity that may be more closely related to human stem cells than to early T ll
T‐cell precursors; suggesting it is more likely a stem cell rather than ti it i
lik l
t
ll th th
a T cell precursor • Molecular genetics
Molecular genetics
• Increase in AML‐associated mutations
• Rare NOTCH pathway (T‐ALL‐associated) mutations
• Considered high riskCoustan‐Smith E, et al. Lancet Oncol 10:147, 2009
Haydu JE and Ferrando AA. Curr Opin Hematol 20:369, 2013
Al
Algorithmic
ith i Approach
A
h to
t AL
Morphologic Review
Immunophenotype
Cytogenetics
Cytogenetics in AL
• AL cytogenetic risk groups are welldefined
Moorman AV, et al. Blood 109:3189, 2007
Grimwade D, et al. Blood 116:343, 2010
Cytogenetics of AML ‐ Overall Survival
10
1.0
0.9
0.8
Survvival Distribu
ution Function
n
0.7
0.6
Low risk (n=69)
0.5
0.4
Intermediate risk (n=98)
Intermediate risk (n
98)
03
0.3
0.2
Hi h i k ( 62)
High risk (n=62)
0.1
p < 0.0001
0.0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
Overall Survival (mo.)
Arber et al Am J Clin Pathol 119:672, 2003
Cytogenetics in AL
• AL cytogenetic risk groups are welldefined
• Some karyotype abnormalities define
specific disease entities
WHO Classification of Precursor Myeloid and Lymphoid Neoplasms (4th Edition)
Acute myeloid leukemia (AML) and related precursor neoplasms
y
(
)
p
p
•
AML with recurrent genetic abnormalities
– AML with t(8;21) (q22;q22) (RUNX1‐RUNX1T1)
– AML with inv(16)(p13.1q22) or t(16,16) (p13.1;q22) (CBFB‐
MYH11)
– Acute promyelocytic leukemia with t(15;17)(q24.1;q21.1) (PML‐RARA)
– AML with t(9;11)(p22;q23) (MLLT3‐MLL)
– AML with t(6;9)(p23;q34) (DEK‐NUP214)
– AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) (RPN1
AML with inv(3)(q21q26 2) or t(3;3)(q21;q26 2) (RPN1‐
EVI1)
– AML (megakaryoblastic) with t(1;22)(p13;q13) (RBM15‐
MKL1)
– Provisional entity: AML with mutated NPM1
– Provisional entity: AML with mutated CEBPA
•
AML with myelodysplasia‐related changes
•
Therapy‐related myeloid neoplasms •
AML not otherwise specified
– AML minimally differentiated
AML minimally differentiated
– AML without maturation
– AML with maturation
– Acute myelomonocytic leukemia
– Acute monoblastic and monocytic leukemia
y
– Acute erythroid leukemia
– Acute megakaryocytic leukemia
– Acute basophilic leukemia
– Acute panmyelosis with myelofibrosis
•
Myeloid sarcoma
Myeloid sarcoma
•
Myeloid proliferations related to Down syndrome
•
Blastic plasmacytoid dendritic cell neoplasm
Acute leukemias of ambiguous lineage
•
Acute undifferentiated leukemia
Acute undifferentiated leukemia
•
Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); BCR‐
ABL1
•
Mixed phenotype acute leukemia with t(v;11q23); MLL rearranged
•
Mixed phenotype acute leukemia, B/myeloid, NOS
•
Mixed phenotype acute leukemia, T/myeloid, NOS
•
Mixed phenotype acute leukemia, NOS, rare types
•
Other ambiguous lineage leukemias
Precursor lymphoid neoplasms
•
Bl
B‐lymphoblastic leukemia/lymphoma, not otherwise specified
h bl ti l k i /l
h
t th
i
ifi d
•
B‐lymphoblastic leukemia/lymphoma with t(v;11q23)(MLL)
•
B‐lymphoblastic leukemia/lymphoma with t(12;21)(p13;q22) (ETV6‐
RUNX1)
•
y p
/y p
( ; )(q ;q ) (
B‐lymphoblastic leukemia/lymphoma with t(5;14)(q31;q32) (IL3‐
IGH@)
•
B‐lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3) (TCF3‐PBX1)
•
B‐lymphoblastic leukemia/lymphoma with hyperdiploidy
•
Bl
B‐lymphoblastic leukemia/lymphoma with hypodiploidy
h bl ti l k i /l
h
ith h
di l id
•
T‐lymphoblastic leukemia/lymphoma
Cytogenetics in AL
• AL cytogenetic risk groups are welldefined
• Some karyotype abnormalities define
specific disease entities
• Myelodysplasia-related cytogenetic
abnormalities are a key feature for a
diagnosis of AML with myelodysplasia
myelodysplasiarelated changes
MDS-related cytogenetic
abnormalities
• C
Complex
l k
karyotype*
t
*
• Unbalanced
abnormalities
–
–
–
–
–
–
–
–
-7/del(7q)
-5/del(5q)
i(17q)/t(17p)
-13/del(13q)
del(11q)
del(12p)/t(12p)
del(9q)
idic(X)(q13)
• B
Balanced
l
d
abnormalities
–
–
–
–
–
–
–
–
–
t(11;16)(q23;p13.3)
t(11;16)(q23;p13.3)**
t(3;21)(q26.2;q22.1)**
t(1;3)(p36.3;q21.1)
t(2 11)( 21 23)**
t(2;11)(p21;q23)**
t(5;12)(q33;p12)
t(5;7)(q33;q11.2)
( )(q q
)
t(5;17)(q33;p13)
t(5;10)(q33;q21)
t(3;5)(q25;q34)
*>3 abnormalities ** must exclude therapy‐related disease
Cytogenetics in AL
• AL cytogenetic
t
ti risk
i k groups are well-defined
ll d fi d
• Some karyotype abnormalities define
specific disease entities
• Myelodysplasia-related
y
yp
cytogenetic
y g
abnormalities are a key feature for a
diagnosis
g
of AML with myelodysplasiay
yp
related changes
• A normal karyotype
y yp is the trigger
gg for
additional molecular genetic testing in
some situations ((Question 4))
KEY QUESTION 3: AT THE TIME OF DIAGNOSIS, WHAT
TESTS ARE REQUIRED FOR ALL PATIENTS FOR THE INITIAL
EVALUATION OF AN ACUTE LEUKEMIA?
Essential
i. Morphologic assessment (blood and bone
marrow)
ii. Conventional cytogenetic analysis
( y yp )
(karyotype)
iii. Flow cytometric immunophenotyping with
a panel sufficient to recognize/distinguish
AML (including APL), T-ALL (including ETPALL), B-ALL, and MPAL, to provide a
baseline for future MRD analysis and to
identify possible therapeutic targets**
KEY QUESTION 3: AT THE TIME OF DIAGNOSIS, WHAT
TESTS ARE REQUIRED FOR ALL PATIENTS FOR THE INITIAL
EVALUATION OF AN ACUTE LEUKEMIA?
** [Footnote text: When sufficient bone marrow
aspirate
i
or peripheral
i
blood material
i iis not available
i
for flow cytometry immunophenotyping, such as in
patients with pancytopenia and an inaspirable
marrow due to fibrosis (“dry-tap”),
immunohistochemical studies may be a useful
alternative method for performing limited
immunohistochemical studies.]
KEY QUESTION 3: AT THE TIME OF DIAGNOSIS, WHAT
TESTS ARE REQUIRED FOR ALL PATIENTS FOR THE INITIAL
EVALUATION OF AN ACUTE LEUKEMIA?
May be useful
i. AML
i. Cytochemical
studies
i. MPO or Sudan
S d
black B, nonspecific esterase
or combined
esterase
(Napthol-ASDchloroacetate
esterase + nonnon
specific esterase)
Key Question 4
Which tests should be performed only on
a subset of p
patients, including
g in response
p
to results of initial tests and morphology?
WHO Cl
Classification
ifi ti
• Precursor lymphoid
neoplasms
– B-lymphoblastic
leukemia/
lymphoma NOS
lymphoma,
– B-lymphoblastic
leukemia// lymphoma
y p
with recurrent
genetic abnormalities
– T-lymphoblastic
Tl
h bl ti
leukemia/ lymphoma
B Lymphoblastic Leukemia/Lymphoma
with Recurrent Genetic Abnormalities
• With t(9;22)(q34;q11.2);
(
)(q q
) BCR-ABL1
• With t(v;11q23); MLL rearrangement
(
)(p q ) TEL-AML1 ((ETV6• With t(12;21)(p13;q22);
RUNX1)
• With hyperdiploidy
• With hypodiploidy
• With t(5;14)(q31;q32); IL3-IGH
• With t(1;19)(p23;p13.3); E2A-PBX1 (TCF3PBX1)
Genetics of ALL
t(12;21)
(
) ETV6-RUNX1
t(9;22) BCR-ABL1
t(1;19) TCF3-PBX1
11q23 MLL
t(5;14) IL3-IGH
Hyperdiploid
Hypodiploid
Adult
<2%
20-30%
3%
3-6%
<1%
<2%
1-5%
Peds
20-25%
2-4%
5-6%
2-3%
<1%
25%
1-5%
Precursor B-cell
B cell ALL Prognosis
Favorable
ALL with t(12;21)
Hyperdiploidy
Unfavorable
ALL with t(9;22)
ALL with t(4;11)
ALL with t(1;19)
Hypodiploidy
ALL with t(9;22)(q34;q11.2);
t(9;22)(q34;q11 2); BCR-ABL1
BCR ABL1
• More common in adults
• Usually precursor B (CD19,
(CD19 CD10 and
TDT+) with CD13, CD33, CD38 and CD25
common
• Rare precursor T cases
• Poor prognosis
• Followed
Follo ed b
by q
quantitative
antitati e PCR for BCRBCR
ABL1
ALL with t(12;21)(p13;q22); TEL
TELAML1 (ETV6-RUNX1)
• More common in children
• Not identified by karyotype
• Precursor B with bright CD10,
CD10 often CD13
and CD34 positive
• Good prognosis
• Late relapses
Other Abnormalities
• B-lymphoblastic leukemia
– Mutations/deletions
o
o
o
o
PAX5
CDKN2A/B
IKZF1 (IKAROS)
JAK1, JAK2, JAK3
– Translocations
o CRLF2
– Amplification
o iAMP21
PAX5 Mutations
• One of several genes mutated that
encode regulators of B cell
development and differentiation
• PAX5 located at 9p13
p
• Detected in 31.7% of B-ALL cases,
g most genetic
g
subtypes
yp
involving
• Mutations results in decreased PAX5
protein expression
• Methylation of PAX5 not detected
• No p
prognostic
g
significance
g
Mullighan et al. Nature 446;758, 2007
IKZF1 Deletions
• IKZF1 at 7p12
encodes the zinc
finger transcription
factor IKAROS
• Deleted in 84% of
PH+ ALL and
deletions acquired
with lymphoid blast
crisis of CML
• IKZF1 deletions
frequently
associated with
deletions of
CDKN2A and/or
PAX5
Mullighan et al. Nature 453;110, 2008
IKZF1 Deletions
• Detected in 28.6%
of pediatric Phnegative ALL
• Absent in ALL with
t(12;21)
• Associated with
gene expression
signature similar to
Ph+ ALL
• Very poor prognosis
independent of
age, WBC count
and genetic
subtype
Mullighan et al. N Engl J Med 360;470, 2009
JAK Mutations
• Known association
with MPNs, Down
syndrome associated
B-ALL, and T-ALL
• JAK1, JAK2 and JAK3
mutations found in
10.7% of Ph-negative
B-ALL (80% JAK2)
• Mutations associated
with deletion of IKZF1
and CDKN2A/B and a
Ph+ ALL gene
expression profile
• Very poor prognosis of
IKZF1 deleted/JAK
mutated cases
Mullighan et al. PNAS 106;9414, 2009
CRLF2 Translocations
• Found in 7-14% of B-ALLs and in 53% of
Down-syndrome associated ALL
• Located at Xp22.3/Yp11.3
• 62% are translocations with IGH
• Associated with
–
–
–
–
JAK1 and JAK2 mutations
IKZF1 deletions
Hispanic ethnicity
Very poor prognosis
Mullighan et al. Nat Genet 41:1243, 2009
Harvey et al. Blood 115:5312, 2010
BCR ABL1 like B-ALL
BCR-ABL1-like
B ALL
• BCR-ABL1-like B-ALL is a high
g risk ALL with a
gene expression profile similar to that of BCRABL1+ ALL, and is characterized by alterations
g
g
in cytokine receptors and kinase signaling
genes leading to activated kinase signaling
pathways.
• Accounts for 10% of pediatric and 25% of
adult ALL; poor clinical outcomes; may be
amenable to targeted therapy.
• Need to establish clear diagnostic criteria
• About
Ab t 50% or more cases may b
be
detected by flow cytometry analysis of
CRLF2
• Some
S
h
have activating
ti ti
mutations
t ti
or
rearrangements of genes, such as ABL1,
JAK2, PDGFRB, CRLF2, EPOR, and/or of
IKZF1 deletions/mutations.
deletions/mutations
• The full spectrum of genetic changes is still
van der Veer et al.
being investigated.
Blood 122:2622, 2013
Izraeli. Curr Opin Hematol 21:289, 2014
ALL with iAMP21
• Intrachromosomal amplification of
chromosome 21 (iAMP21) accounts for
about 2% of B-ALL
• Generally in older children (median age
9 years) with lower WBC count
• Adverse outcomes when treated with
standard risk therapy; but improved
when treated as high risk ALL
• Presence
ese ce o
of 4-5
5o
or more
o e cop
copies
es o
of RUNX1
U
on a single chromosome.
• Reliably detected by FISH for RUNX1 and
confirmed by
y cytogenetics.
y g
• May have some overlap with BCR-ABL1like B-ALL.
Image provided by Dana Bangs
Harrison et al. Leukemia 28:1015, 2014
Other Abnormalities
• T-lymphoblastic leukemia
– Translocations involving
o
o
o
o
o
o
TRB (7q32)
(7 32)
TRA (14q11)
TRD (14q11)
TLX1 (favorable prognosis)
TLX3
TAL1
– Mutations/deletions
o NOTCH pathway (favorable prognosis in
children)
– NOTCH1 (50-60%)
– FBXW7 (10
(10-20%)
20%)
o FLT3 (4%)
Minimal Residual Disease (MRD)
Testing in ALL
• Molecular
M l
l genetic
ti (PCR) approach
h
– Translocations
o Not present in all patients
o Variable quantitation
– Antigen
ge receptor
ecep o rearrangements
ea a ge e s
o Labor intensive
o May detect 0.001 to 0.0001% disease
• Immunophenotype (flow
(f
cytometry)) approach
– Usually involved detection of differential expression
of markers such as CD58
CD58, CD81
CD81, CD123,
CD123 CD304,
CD304
others
– May detect 0.01
0.01-0.001%
0.001% disease
Reviewed by Campana D. Curr Opin Hematol 19:313, 2012
MRD testing in ALL
• MRD is a better predictor of outcome
than WBC count, age, genetic type or
early response to prednisone
• Five-year
Fi
eventt ffree survival
i lb
based
d on
MRD on days 33 and 78
– 92.3% when less than 0.01% for both (42%)
– 77.6%
77 6% when less than 0
0.1%
1% on day 78 (6%)
– 50.1% when 0.1% or greater at day 78 (52%)
Conter V et al. Blood 115:3206, 2010
KEY QUESTION 4: WHICH TESTS SHOULD BE PERFORMED
ONLY ON A SUBSET OF PATIENTS
PATIENTS, INCLUDING IN
RESPONSE TO RESULTS OF INITIAL TESTS AND
MORPHOLOGY?
Patients with ALL:
Essential
i. CSF for cell count and morphology
ii FISH or PCR:
ii.
i. Pediatric B-ALL***:
i.
ii.
iii.
iv.
v.
t(12;21)(p13;q22); ETV6-RUNX1
t(9;22)(q34;q11.2); BCR-ABL1
i. Q‐PCR for patients with confirmed BCR‐ABL1 B‐ALL
MLL translocations
iAMP 21
Trisomy 4 and 10 (FISH or CGH/SNP microarray)
ii. Adult B-ALL*** :
i.
t(9;22)(q34;q11.2);
t(9
22)( 34 11 2) BCR-ABL1
BCR ABL1
i. Q‐PCR for patients with confirmed BCR‐ABL1 B‐ALL
KEY QUESTION 4: WHICH TESTS SHOULD BE PERFORMED
ONLY ON A SUBSET OF PATIENTS
PATIENTS, INCLUDING IN
RESPONSE TO RESULTS OF INITIAL TESTS AND
MORPHOLOGY?
Patients with ALL:
*** [Footnote text: FISH for MYC translocations
may be helpful in excluding cases of Burkitt
leukemia/lymphoma with ambiguous flow
cytometry studies, but are not included in
the guideline because Burkitt
leukemia/lymphoma is no longer
categorized as an acute leukemia.]
leukemia ]
KEY QUESTION 4: WHICH TESTS SHOULD BE PERFORMED
ONLY ON A SUBSET OF PATIENTS
PATIENTS, INCLUDING IN
RESPONSE TO RESULTS OF INITIAL TESTS AND
MORPHOLOGY?
Patients with ALL:
Essential
iii. Flow cytometry panel/analysis or molecular
characterization sufficient to allow subsequent
detection of 0.01% MRD
KEY QUESTION 4: WHICH TESTS SHOULD BE PERFORMED
ONLY ON A SUBSET OF PATIENTS
PATIENTS, INCLUDING IN
RESPONSE TO RESULTS OF INITIAL TESTS AND
MORPHOLOGY?
Patients with ALL:
Recommended
i. IKZF1 deletions
ii CRLF2 translocations
ii.
May be useful
i. CSF by flow
ii. Mutational analysis
i. B-ALL: PAX5, JAK1/2
ii. T-ALL: NOTCH1, FBXW7
© 2014 College of American Pathologists. All rights reserved.
112
2008 WHO Classification of AML
• Acute myeloid leukemia (AML) with
recurrent genetic abnormalities
• AML with myelodysplasia-related
changes
h
• Therapy-related myeloid neoplasms
• AML, not otherwise specified
• Myeloid proliferations related to
Down syndrome
2008 WHO Classification of AML
•
AML with recurrent genetic
abnormalities
– AML with t(8;21) (q22;q22)
(RUNX1-RUNX1T1)
– AML with inv(16)(p13.1q22)
( )(p
q ) or
t(16,16) (p13.1;q22) (CBFB-MYH11)
– Acute promyelocytic leukemia
with t(15;17)(q24.1;q21.1) (PMLRARA)
– AML with t(9;11)(p22;q23) (MLLT3MLL)
– AML with t(6;9)(p23;q34) (DEK(DEK
NUP214)
– AML with inv(3)(q21q26.2) or
t(3;3)(q21;q26 2) (RPN1-EVI1)
t(3;3)(q21;q26.2)
(RPN1 EVI1)
– AML (megakaryoblastic) with
t(1;22)(p13;q13) (RBM15-MKL1)
– Provisional
o s o a entity:
e y: AML with
mutated NPM1
– Provisional entity: AML with
mutated CEBPA
•
•
•
•
•
•
AML with myelodysplasia-related
changes
Therapy-related myeloid neoplasms
AML, not otherwise specified
– AML minimally differentiated
– AML without maturation
– AML with maturation
– Acute myelomonocytic leukemia
– Acute monoblastic and
monocytic leukemia
– Acute erythroid leukemia
– Acute megakaryocytic leukemia
– Acute basophilic leukemia
– Acute panmyelosis with
myelofibrosis
Myeloid sarcoma
Myeloid proliferations related to Down
syndrome
Blastic plasmacytoid dendritic cell
neoplasm
Acute promyelocytic
p
y
y leukemia with
t(15;17)(q24.1;q12); PML-RARA
• High rate of disseminated intravascular coagulopathy
• Good prognosis when treated with regimens that include
ATRA
• Subset have mutations of FLT3-ITD, which confers a worse
prognosis in adults
Core Binding Factor Leukemia
AML with t(8;21) (q22;q22) (RUNX1‐RUNX1T1)
(RUNX1
RUNX1T1)
AML with inv(16)(p13.1q22) or t(16;16)(p13 1;q22); CBFB‐
t(16;16)(p13.1;q22); CBFB
MYH11
KIT Mutations
• Mutations occur in 22-30% of
AMLs with t(8;21) and inv(16)
• Located at 4q11-12
• Type III tyrosine kinase that
encodes a 145-kD
transmembrane glycoprotein
• Ga
Gain o
of function
u c o mutations
ua o s
occur in
o
o
o
o
GIST
Germ cell tumors
Mastocytosis
AML
• In AML, mutations involve
exon 17 (usually D816V) or
exon 8
• Mutations in core binding
factor AMLs associated with a
worse prognosis
Paschka et al. J Clin Oncol 24:3904, 2006
Gotlib J. Immunol Clin N Am 26:575, 2006
AML with mutated NPM1
• 25
25-30%
30% off allll AML
AMLs; 47
4762% with normal
karyotypes
• Women, high WBC and
Plt counts
• Wild (non-mutated)
NPM1 associated with
worse prognosis
• NPM1+/FLT3- good
prognosis
• Provisional entity in
2008 WHO
Thiede et al. Blood 107:4011, 2006
AML with mutated NPM1
• NPM1 located at 5q35
• Typically heterozygous
mutations occurring at
exon 12
• Exon 9 and 11 mutations
less common
• Mutations alter C-terminus
C terminus
of the protein, which
mediates aberrant
locali ation of NPM to the
localization
cytoplasm
• Wild type
yp NPM localized
to the nucleus
• Immunohistochemical
detection of NPM in
cytoplasm correlates with
mutation
AML with mutated CEPBA
• Also known as CCAAT/enhancer binding
protein-α
9q 3
• Located at 19q13.1
• Wild type encodes a transcription factor
that controls myeloid
y
p
progenitor
g
cell
proliferation and differentiation
• Mutations occur in 7-20% of AMLs
• Most frequent with normal or intermediate
karyotype
• Homozygous/biallelic mutations
associated with a favorable prognosis
• Provisional
ii
entity
i iin 2008 WHO
Preudhomme et al. Blood 100:2717, 2002
FLT3 Mutations
• FLT3 located at 13q12
• Encodes a tyrosine kinase receptor
involved in hematopoietic stem cell
differentiation and proliferation
• Two types of mutations
• Internal tandem duplication (FLT3-ITD)
•
•
Occur within the juxtamembrane domain
Represent 75-80% of mutations
• Tyrosine
T
i
kinase
ki
domain
d
i mutations
t ti
(FLT3(FLT3
TKD)
•
•
Codons 835 or 836 of the second tyrosine
kinase domain
Represent 20-35% of mutations
• Mutations occur in 10-15% of
childhood AMLs and 20-28% of adult
y yp
AMLs; 25-30% with normal karyotypes
• More frequent in APL, normal
karyotype AML or t(6;9) AML
• ITD mutation associated with
decreased disease free survival in
adults
Kottaridis et al. Blood 98:1752, 2001
Mutations in AML
Gene
Frequency in AML
Reported prognosis
NPM1
FLT3 ITD
DNMT3A
NRAS/KRAS
WT1
RUNX1
IDH2 R132
IDH2 R140 and R172
TET2
MLL
ASXL1
FLT3 TKD
CEBPA
PTPN11
PHF6
TP53
KIT
CBL
EZH2
JAK2
30-35%
25%
15-25%
15-20%
10-15%
10-15%
7-16%
88-15%
15%
8-12%
5-10%
3-19%
7%
6%
3%
2-4%
2-5%
2-3%
1-3%
1-3%
1%
Favorable
Unfavorable
Unfavorable
Neutral
Neutral to unfavorable
Unfavorable
Variable
Variable
Unfavorable
Unfavorable
Unfavorable
Neutral
Favorable
Unknown
Unfavorable
Unfavorable
Unfavorable
Unknown
Unknown
Unfavorable
Mutations in AML
• Different classes of
mutations occur in
AML
• Some impact the
epigenetics of
AML
Bravo et al. Br J Haematol ePub June 5, 2014
AML with mutated RUNX1
• Gene located at 21q22
• Encodes the alpha subunit of
the core binding factor
• Mutation in 12.5-13.2% of AML
• More frequent in older male
p
patients
• Frequent prior history of MDS, or
prior exposure to radiation
• Immature morphology and
phenotype
• Frequent associated MLL-PTD
or ASXL1 mutations
• Rare CEBPA or NPM1 mutations
• Poor response to therapy with
shortened survival
Tang et al. Blood 114:5352, 2009
Mendler et al. J Clin Oncol 30:3109, 2012
Epigenetics and AML
• Protein expression
– ERG,
G, BAALC,
C, EVI1
• Methylation
– Global
Gl b l
– Gene
• Histone modifiers
• RNA splicing
GSTM1 methylation
Ohgami et al Br J Haematol 159;182, 2012
Ohgami et al. Br J Haematol
159;182 2012
Epigenetics and Myeloid Neoplasms
• Protein expression
– ERG,
G, BAALC,
C, EVI1
• Methylation
– Global
Gl b l
– Gene
• Histone modifiers
• RNA splicing
Bravo et al. Br J Haematol ePub June 5, 2014
KEY QUESTION 4: WHICH TESTS SHOULD BE PERFORMED
ONLY ON A SUBSET OF PATIENTS
PATIENTS, INCLUDING IN
RESPONSE TO RESULTS OF INITIAL TESTS AND
MORPHOLOGY?
Patients with AML:
Essential
i. Rapid detection of PML-RARA from blood or
marrow if acute promyelocytic leukemia is
morphologically, phenotypically or clinically
suspected
ii. Mutational analysis:
i. For core binding
g factor AMLs:
i. KIT
ii. For acute promyelocytic leukemia:
i. FLT3-ITD
iii. For AML with myelodysplasia- related cytogenetic
abnormalities:
i. none
i For
iv.
F allll other
th AML types
t
(outside
( t id off the
th above
b
groups):
)
i. NPM1, FLT3-ITD, CEBPA
KEY QUESTION 4: WHICH TESTS SHOULD BE PERFORMED
ONLY ON A SUBSET OF PATIENTS
PATIENTS, INCLUDING IN
RESPONSE TO RESULTS OF INITIAL TESTS AND
MORPHOLOGY?
Patients with AML:
Recommended
i. CSF for cell count and morphology for patients
with CNS symptoms or signs
ii Mutational analysis:
ii.
i. IDH1, IDH2, TET2, RUNX1, WT1, DNMT3A
iii. Gene expression analysis:
i. ERG, BAALC, EVI1
May be useful
i. Global/gene specific methylation
ii. miRNA expression
p
iii. Targeted FISH panels
Acute Leukemias of Ambiguous
Lineage
• A
Acute
t undifferentiated
diff
ti t d leukemia
l k
i
• Mixed phenotype acute leukemia
with t(9;22)(q34;q11.2);
t(9;22)(q34;q11 2); BCR-ABL1
BCR ABL1
• Mixed phenotype acute leukemia
with t(v;11q23); MLL rearranged
• Mixed phenotype acute leukemia,
B/myeloid NOS
B/myeloid,
• Mixed phenotype acute leukemia,
T/myeloid NOS
T/myeloid,
• Mixed phenotype acute leukemia,
NOS rare types
NOS,
• Other ambiguous lineage leukemias
KEY QUESTION 4: WHICH TESTS SHOULD BE PERFORMED
ONLY ON A SUBSET OF PATIENTS
PATIENTS, INCLUDING IN
RESPONSE TO RESULTS OF INITIAL TESTS AND
MORPHOLOGY?
For patients with MPAL:
Recommended
i.i FISH or PCR:
i. t(9;22)(q34;q11.2); BCR-ABL1
i.i Q
Q-PCR
PCR for patients with confirmed BCR
BCR-ABL1
ABL1 B
B-ALL
ALL
ii. MLL translocations
KEY QUESTION 5. WHERE SHOULD
TESTING BE PERFORMED?
Purpose off thi
this question:
ti
1) Assure testing is done by accredited, reliable laboratories
2)) To minimize duplicate
p
and p
particularly
y invasive p
procedures
if the patient is transferred to a tertiary care center
3) To assure data is transferred appropriately from the initial
institution where the patient was initially diagnosed with
acute leukemia to the treating institution
4) To assure that any specimen preserved from the initial
i tit ti
institution
th
thatt may be
b valuable
l bl iin the
th diagnosis,
di
i
classification, and prognosis of the leukemia be
patient’s p
permission, to the
transferred, with the p
appropriate institutions for assessment
5) To aid in cost containment
© 2014 College of American
Pathologists. All rights reserved.
131
KEY QUESTION 5. WHERE SHOULD
TESTING BE PERFORMED?
i.i All laboratory testing performed for the initial work-up
work up and
diagnosis of a patient suspected to have acute leukemia should
be performed in a laboratory that is CLIA or CAP accredited (or
international equivalent) for high complexity testing procedures.
i. Sufficient clinical information pertinent to the evaluation
should be provided to the testing personnel to allow for
appropriate triaging of the sample in the testing laboratory
and for proper interpretation of the results (see Key Question
1 a, ii)
1.
ii).
ii. Tests performed for classification, management, predicting
prognosis, and monitoring the response to therapy should ideally
be coordinated by the institution where the patient will be
treated, and in laboratories in those institutions that meet the
requirements stated in 5i.
5i
© 2014 College of American
Pathologists. All rights reserved.
132
KEY QUESTION 5. WHERE SHOULD
TESTING BE PERFORMED?
iii. In order to limit the number of invasive p
procedures for the p
patient
yet obtain the appropriate testing for optimal patient
management, as well as for the purposes of cost containment, it is
recommended that invasive procedures
procedures, including bone marrow
aspirations and biopsies, consider deferring to the treatment
center whenever possible. Alternatively, fresh or cryopreserved
cells from an invasive procedure,
procedure such as bone marrow aspiration
aspiration,
may be sent to the tertiary treatment center at or before the time
of transfer
iv. All laboratory reports/results and pathology slides of all testing
performed at the primary institution and a list of pending tests
patient referral to the
should be made available at the time of p
tertiary center to confirm the diagnosis.
i. In the case of flow cytometry, scatter plots and list mode data
should be made available if requested.
requested
ii. In the case of cytogenetics, original karyograms should be
133
provided if requested.
Key Question 6
How should test results and the diagnosis
be correlated and reported?
p
Key Elements for a Pathologic
Diagnosis
• Clinical information
• Morphology
• Immunophenotyping
– Immunohistochemistry
– Flow cytometry
• Cytogenetics
y g
– Karyotype
– FISH
• Molecular genetics
– Various techniques
The Bone Marrow Report (CAP
Guidelines)
•
•
•
•
•
•
•
•
•
•
Clinical information
Aspirate and biopsy
sites
Peripheral blood
Marrow
aspirate/touch
i t /t
h preps
Marrow biopsy/clot
Immunophenotyping
Cytogenetics
Molecular genetics
Other ancillary tests
Diagnosis
Why Comprehensive Reporting
Matters
• Yesterday
– French-American-British
e c
e ca
s
(FAB) Classification
o Morphology
o Cytochemistry
o A little
immunophenotyping
o No genetics
g
o Little clinical relevance
Why Comprehensive Reporting
Matters
• Yesterday
• Today
– World Health
Organization (WHO)
Classification
o
o
o
o
o
Clinical information
Morphology
Immunophenotyping
C t
Cytogenetics
ti
Some molecular genetics
Why Comprehensive Reporting
Matters
• Yesterday
Y t d
• Today
–W
World
ld H
Health
lth
Organization (WHO)
C ass ca o
Classification
o
o
o
o
o
Clinical information
Morphology
Immunophenotyping
Cytogenetics
Some molecular genetics
• Tomorrow
o Explosion of molecular
genetics
How are we going to report all of
these changes?
• Challenges
– Technology
g
o Laboratory information
systems tend to be
d i
designed
d tto create
t
separate reports
o Many hospitals use different
information systems for
different laboratories
o Hospital information
systems/electronic medical
records do not tend to
display data in a readable
format
CBC Data
S
Sunquest
BM Report
PowerPath
• Stanford approach
– Partially interfaced
– Mostly manual
Flow Cytometry
l C
Initial Report Issued
FISH Results
PowerPath
Amended Report Issued
Molecular Report
p
Sunquest
Amended Report Issued
Karyotype Results
Amended Report Issued
Amended Report Issued
Comprehensive Reporting
• Stanford approach
• Vanderbilt approach
– Thanks
Th k to
t Mary
M
Z tt
Zutter
Vanderbilt use of integrated reporting and
decision support tools
1
Clinical context is provided Clinical
context is provided
through electronic bone marrow testing form
2
Created algorithms to organize test recommendations
Created algorithms to organize test recommendations
Disease
A. Diagnosis
1. ALL
A1
2. AML/MDS
A2
3. Lymphoma
A3
4. BM Failure
A4
5. MPN
5 MPN
A5
6. Myeloma
A6
B. Staging
C. Follow‐
up
C1
A2: AML/MDS ‐ Diagnosis
o Flow Cytometry
Flow Cytometry
o Cytogenetics C2
• Karyotype
B3• MDS FISH Panel
C3
AML FISH Panel
• AML FISH Panel
o Molecular
C4
• NPM1
• CEBPA
C5
• FLT3
• c‐Kit
C6
Vanderbilt designed dashboards with indicators to help communicate testing status, improving workflow in the lab
i t t ti
t t i
i
kfl i th l b
Status indicators:
v = pending
green = all tests in category
resulted
yellow = some resulted,
some pending
Dashboard also provides secure
messaging
Vanderbilt created integrated, comprehensive reports to allow the clinician to quickly correlate & apply results
allow the clinician to quickly correlate & apply results
Comprehensive diagnosis Comprehensive
diagnosis
accounting for all test results
Personalized prognostic/ P
li d
ti /
therapeutic information
Report fields automatically populated
automatically populated by data from individual pathology reports
Benefits of Comprehensive Benefits of Comprehensive Report • Consistent format
• Integration of multiple reports
I t
ti
f
lti l
t
• Structured reporting to enable clinical decision support & research
pp
How are we going to report all of
these changes?
• Challenges
– Technology
ec o ogy
– Apathy
KEY QUESTION 6: HOW SHOULD TEST RESULTS AND THE
DIAGNOSIS BE CORRELATED AND REPORTED?
i. All test results, including those performed by
referral laboratories
laboratories, must appear on the
patient’s medical record. The results must
indicate the source of the sample and its
adequacy,
d
ti
time/day
/d off collection,
ll ti
ti
time/day
/d
and location of testing, and time/day of the
report.
ii. Because testing that is necessary for
g
classification, predicting
p
g prognosis
p g
diagnosis,
and monitoring the response to therapy is
frequently performed in various laboratories, a
mechanism should be in place to coordinate
the results, including those from outside
reference laboratories. The pathologist should
play
l th
the central
t l role
l in
i coordination
di ti
off this
thi
data in a single written report.
KEY QUESTION 6: HOW SHOULD TEST RESULTS AND THE
DIAGNOSIS BE CORRELATED AND REPORTED?
iii. The WHO classification scheme is recommended for the
classification of acute leukemia. The criteria/rationale for reaching
the final WHO classification/diagnosis should be clearly summarized
and outlined in the final report and should also include
i.
Pertinent clinical data, such as prior therapy, predisposing diseases, etc.
ii
ii.
Cellularity of bone marrow
marrow, percentage of blasts in blood and bone
marrow, pattern of maturation/myelodysplasia, if any, in the
hematopoietic lineages
iii.
Specific/unique morphologic features of the leukemia helpful for
classification
l ifi ti
and
d recognition
iti
off the
th neoplastic
l ti cells
ll in
i follow-up
f ll
specimens, e.g., abnormal eosinophils, Auer rods, etc.
iv. The presence of any additional findings of importance, i.e., co-existing
tumor, marked hemophagocytosis,
p g y
or the p
presence of necrosis or fibrosis,
etc.
v.
A summary of the phenotype of the leukemic cells as determined by
multiparameter flow cytometry, or, in the absence of sufficient aspirated
cells by flow cytometry, the results of additional cytochemical or
immunohistochemical stains.
i.
A summary of the results of genetic studies, including karyotyping,
FISH, and molecular genetic studies.
ii.
A summary of specific features that may be used for monitoring the
disease in subsequent specimens should be included.
KEY QUESTION 6: HOW SHOULD TEST RESULTS AND THE
DIAGNOSIS BE CORRELATED AND REPORTED?
iv.Although all data may not be available
simultaneously, an initial report with the
essential morphologic and
immunophenotypic data with a list of
known pending tests can still be issued to
provide as much information as possible,
with additional addendum/amended
reports as additional data becomes
available.
How will the guidelines relate to any
WHO revision?
Revision of the 4th edition
• C
Currentt “blue
“bl
b
book”
k” iis partt off th
the 4th
edition series, starting in 2008
• Needs updating, but WHO has not
completed
p
all 4th edition books and will
not allow 5th edition to begin until entire
4th edition series is complete
• Will allow an on-line revision of the 4th
edition
• Currently no plans for a hard copy
Clinical Advisory Committee
• March
M
h 31 and
d April
A il 1,
1 2014
2014, Chicago,
Chi
IL
– Organized by Jim Vardiman and Michelle LeBeau
– 50 invited
i it d participants
ti i
t ((pathologists,
th l i t
cytogeneticists, hematologists) and for acute
leukemia and myeloid
y
neoplasms
p
topics
p
– 50 invited participants for lymphoid neoplasms
• Acute Leukemia and Myeloid
y
Neoplasms
p
CAC
Co-Chairs Clara Bloomfield and Mario Cazzola
• A series of q
questions were p
proposed
p
by
y the cochairs and involved pathologists for discussion
and vote by the CAC
Probable WHO Revisions for ALL
• B-ALL
– BCR-ABL1-like
C
e BALL
–B
B-ALL
ALL with iAMP21
– Hypodiploid ALL
will be subdivided
• T-ALL/Other
– Early T-Precursor
ALL
Probable WHO Revisions for AML
• New cytogenetic
subgroups
g
p
– Rare ones being
discussed
– AML with BCR-ABL1
R fi d category
t
– Refined
off
“myeloid neoplasm with
i (3)( 21 26 2)/t(3 3)(
inv(3)(q21q26.2)/t(3;3)(q
21;q26.2)”
– Refine APL with PMLRARA fusion
Probable WHO Revisions for AML
• Ne
New and revised
re ised
mutation subgroups
– AML with RUNX1
mutation
– AML with CEBPA
mutation will have to
be
heterozygous/double
mutation
– NPM1 and CEBPA
mutations will trump
p
multilineage dysplasia
in de novo disease
without MDS-related
cytogenetic
abnormalities
Probable WHO Revisions for AML
• Revise criteria for AML
with myelodysplasiarelated changes
– Remove de novo cases
with no MDS-related
cytogenetic
abnormalities if NPM1 or
CEBPA mutated
• Add section on familial
myeloid neoplasms
– Often associated with
mutations of RUNX1,
GATA2 CEBPA or
GATA2,
ANKRD26
WHO Next Steps
• Editors and reviewers
re ie ers with
ith work
ork with
ith chapter
authors to resolve remaining issues
• Chapter revisions will be drafted
• Proposed changes will be presented to the
y of Hematopathology
p
gy Companion
p
Society
Meeting at the March 2015 US & Canadian
Academy of Pathology Annual Meeting in
Boston for further comment
• Six papers summarizing changes will be
published in Blood
p
• On-line version of complete revision targeted for
late 2015
CAP/ASH Guidelines Next Steps
•
•
•
•
Data extraction correlation
Public comment period
Revision
Publication
Summary/Pearls of Pathology
• R
Reviewed
i
d th
the clinical
li i l iinformation
f
ti
and
d
samples needed to completely evaluate
acute
t leukemia
l k
i samples
l
• Discussed the various ancillary tests
needed for
f all cases and when to order
additional tests on selected cases
• Reviewed the CAP/ASH draft guidelines
and how to use them
• Open comment period soon with revision
and a p
publication target
g date of late 2015

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