ENCCA

Transcription

ENCCA

ENCCA DELIVERABLES
Project no. 261474
ENCCA
EUROPEAN NETWORK for CANCER research in CHILDREN and
ADOLESCENTS
Network of Excellence
Deliverable Number: 9.1
Title:
Common guidelines for diagnostic approaches
to leukemias
Due Delivery Date: M24
Actual Delivery Date: M24
Start date of project: 1 January 2011
Duration: 48 months
Project co-funded by the European Commission within the Seventh Framework Programme
(2011-2014)
Dissemination Level
Public
PU
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Summary
In this deliverable we describe consensus recommendations on the diagnosis of childhood
acute leukemia from a European viewpoint based on surveys of the groups participating to
the AIEOP-BFM ALL 2009 study, a 7-country-based international frontline treatment trial on
acute lymphoblastic leukemia (ALL) in children and adolescents, a survey of the work group
DCOG Molecular Research of the Dutch Childhood Oncology Group, and the Biology &
Diagnosis Committee of the International BFM Study Group, the largest initiative on
childhood leukemias worldwide which unifies cooperative national study groups on
childhood leukemia from more than 30 different, mostly European countries. Topics covered
relate to cytomorphology, immunophenotyping, cyto- and molecular genetics, and the
evaluation of measures of treatment response.
The recommendations provide a common ground for systematic implementation of
molecularly-based diagnostic strategies in acute leukemias to be developed within ENCCA
and, therefore, can be seen as a prerequisite for the implementation of rational molecular
information-based treatment approaches in pediatric hematology and oncology.
The diagnostic guidelines will be published on the website of the international BFM study
group (http://www.bfm-international.org).
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Description
Common guidelines for diagnostic approaches to leukemias
Correspondence to:
Susanne Kilian, PhD, MLaw
University Medical Center Schleswig-Holstein
Department of General Pediatrics ▪ Campus Kiel
Schwanenweg 20 ▪ 24105 Kiel, Germany
Tel.: +49 (0)431 597-3947 ▪ Fax: -3966
Email: [email protected]
Disclaimer:
While the advice and information in these guidelines is believed to be true and accurate,
neither the authors, the International BFM Study Group nor the European Network for
Cancer Research in Children and Adolescents accept any legal responsibility for the
content of this document.
Date of recommendation review:
January 2013
Writing group:
ENCCA WP9 and I-BFM Biology & Diagnosis Committee members
Declarations of Interest:
None of the authors are considered to have a conflict of interest with regards to the
recommendations made in this document.
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Contents
Deliverable in context of WP9
5
Recommendations for the diagnosis of childhood acute lymphoblastic leukemia
General information
Clinical presentation and diagnosis
Prognostic factors and risk-adapted treatment
6
Diagnosis of childhood acute lymphoblastic leukemia
Diagnostic methods
Cytomorphology
Immunophenotyping
Cytogenetics and FISH
Molecular genetics
Treatment response evaluation
Biobanking
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Recommendations for the diagnosis of childhood acute myeloid leukemia
General information
19
Diagnosis of childhood acute myeloid leukemia
Diagnostic methods
Cytomorphology
Immunophenotyping
Molecular genetics
Biobanking
22
References
24
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Deliverable in context of WP9
WP9 aims to establish a harmonized and integrated approach to the rational introduction of
molecularly targeted treatment in clinical trials on leukemias. In order to achieve this
ultimate goal, the development of standardized comprehensive diagnostic approaches as
well as biobanking, and the establishment of a common pipeline for molecular diagnostics
in a European virtual laboratory on leukemias are necessary.
These recommendations are neither obligatory nor is it necessary to follow all of them.
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Recommendations for the diagnosis of childhood acute lymphoblastic
leukemia (ALL)
General information
ALL represents the malignant proliferation of lymphoid cells blocked at an early stage of
differentiation and is the most common malignancy in children. It accounts for
approximately 25% of all childhood cancers and about 80% of childhood leukemias. The
annual incidence rate of childhood ALL varies world-wide between approximately one and
four new cases per 100,000 children younger than 15 years, with a peak incidence at
approximately two to five years of age. More affluent countries tend to have higher
incidence rates. However, incidence rates for childhood ALL do not only vary between
countries, but also by ethnicity within countries: in the USA rates are highest in Hispanic
children and higher in white compared to black children. More than 60% of patients
diagnosed with ALL are children. Treatment results in childhood ALL are one of the true
success stories of clinical oncology with current overall cure rates of approximately 80% in
developed countries. These results are reached by application of intensive multiagent
chemotherapeutic regimens and in specific patient subgroups additional radiotherapy
and/or hematopoietic stem cell transplantation (HSCT). Modern treatment regimens consist
of at least four phases: (1) an induction period aiming at an initial remission induction within
approximately 4 to 6 weeks through the use of multiple cancer chemotherapeutic drugs; (2)
consolidation/intensification and reinduction segments to eradicate residual leukemic blasts
in patients who are in remission by morphologic criteria; (3) extracompartment therapy such
as central nervous system (CNS) preventive therapy, and (4) a maintenance period to
further stabilize remission by suppressing re-emergence of a drug-resistant clone through
continuing reduction of residual leukemic cells. As certain clinically and biologically distinct
patient subgroups with ALL have a particular poor outcome on standard ALL treatment,
clinical protocols specifically addressing the potential therapeutic needs of these subgroups
have been initiated in the recent past (e.g., hybrid protocols for infants, and imatinibincluding regimens for BCR/ABL1-positive ALL).
The initial clinical presentation of a child with ALL largely depends on the extent of the
leukemic infiltration of the bone marrow and extramedullary sites. Typical clinical signs are
fever, pallor, fatigue, bruises, enlargement of liver, spleen and lymph nodes, and pain (e.g.,
bone pain). In most patients, white blood cell counts show anemia, thrombocytopenia and
granulocytopenia with or without concommitant leukocytosis. The diagnosis of ALL is
usually made by cytomorphological and cytochemical examination of a bone marrow
aspirate and in difficult cases by trephine biopsy and is established when at least 25%
lymphoblasts are present in the marrow. CNS involvement (CNS3 status) is diagnosed by
the presence of blasts in the cerebrospinal fluid (CSF; for definition see Table 1) or if
intracerebral infiltrates are detected by cross-sectional radiological imaging. Initial
diagnostics are complemented by flowcytometry-based immunophenotyping to gain
information on the blasts expression of lymphoid differentiation-associated antigens as
measure by the reactivity to specific monoclonal antibodies and to determine the cellular
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DNA content of leukemic cells. In addition, a combined approach using cytogenetic and
molecular genetic techniques is used for the detection of genetic aberrations, such as nonrandom recurrent chromosomal translocations or their molecular equivalents (e.g., the
t(9;22) or the BCR/ABL1 fusion transcript). Molecular-genetic techniques and/or flow
cytometry are also used to monitor disease burden during therapy by measuring minimal
residual disease (MRD). A last important issue addresses the definition of what is called
complete remission and relapse: complete remission is defined as the absence of leukemic
blasts in blood and CSF, fewer than 5% lymphoblasts in bone marrow aspiration smears,
and no evidence of localized disease. Relapse is defined as the recurrence of lymphoblasts
or localized leukemic infiltrates at any site.
Continuing research on the clinical and biological aspects of ALL has identified numerous
features with prognostic potential some of which are displayed in Table 1. On modern
protocols, risk-adapted therapy reflecting the probability of treatment failure has become a
common feature in the clinical management of childhood ALL. For this purpose, the initially
assessed prognostic factors are used to estimate an individual patient’s risk of relapse and
to adjust the required treatment intensity by therapy stratification into different risk groups
(e.g., standard/low, intermediate, high). The prognostic significance of an inadequate early
reduction of leukemic blasts in the peripheral blood was first described by the BFM study
group and confirmed by several other study groups. Of importance, the specificity of
response evaluation might vary with the composition of the induction regimen and the time
point of response evaluation. However, although a poor early response to induction therapy
as described above is highly predictive of treatment failure, the majority of recurrences
occur in the large group of patients with an adequate morphological response to treatment.
Of advantage in this context, the sub-microscopic assessment of MRD is approximately
1000 to 10000-fold more sensitive compared to methods based on morphological detection
and provides excellent prognostic information. Although most of the experience on MRD in
clinical settings was gained through DNA-PCR-based detection of leukemic clone-specific
immunoglobulin and/or T-cell receptor gene rearrangements, it was shown that flowcytometry-based analyses by detection of specific antigen patterns of the leukemic clone
yield sensitive and reliable results comparable to PCR-based methods.
Remission induction treatment
Contemporary treatment approaches for childhood ALL aim at an initial remission induction
and restoration of normal hematopoiesis within approximately 4 to 6 weeks. In most study
groups this goal is achieved in approximately 98% of patients through the systemic
application of three drugs (glucocorticoid, vincristine, L-asparaginase) to which an
anthracycline may be added as a fourth drug. On ALL-BFM protocols, remission induction is
initiated by a 7-day monotherapy with orally administered prednisone (and one intrathecal
dose of intrathecal methotrexate on day 1), which isparticularly useful in avoiding
complications related to extensive tumor cell lysis. Undoubtedly, the dose intensity of the
induction phase can have a major impact on the overall treatment outcome. Nevertheless,
in specific subgroups of childhood ALL, the necessity of a four-drug induction regimen is
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subject to debate and it is, for example, unclear if addition of an anthracycline to a threedrug induction regimen is of real benefit to certain low- or intermediate-risk patients.
Another frequently discussed issue addresses the choice of the glucocorticoid for optimal
induction. Despite some debate on a truly equivalent dose, compared to prednisolone,
dexamethasone appears to have a stronger antileukemic effect in vitro and has been shown
to provide better leukemic CNS control and lower relapse rates. However, dexamethasone
was also associated with increased side effects including severe infectious complications.
The 2% of patients not in remission after induction therapy will either have died of
treatment- or disease-related complications or display nonresponsive disease. The latter
group includes patients that will achieve only delayed remission or show resistant disease.
Because of the poor prognosis of this minor non-responsive patient population, alternative
therapeutic approaches should be considered early during the disease process.
Consolidation/intensification and reinduction treatment
Eradication of residual leukemic blasts in patients who are in remission by morphologic
criteria
is
the
primary
aim
of
consolidation/intensification
treatment.
Consolidation/intensification treatment is necessary as patients successfully induced into
remission, but not given additional treatment, usually relapse within months. A so-called
reinduction or delayed intensification treatment can further enhance the effect of previous
consolidation/intensification therapy. As consolidation/intensification phases administered in
protocols of the large study groups on treatment of childhood ALL are variable and may
differ, for example, with regard to amounts, timing, and number of drug doses, drug
composition and overall treatment context, the direct contributions of most of these
consolidation/intensification strategies and/or their individual components are difficult to
assess and associated with limited generalizability. Today, most protocols use high-dose
methotrexate (combined with folinic acid rescue) together with 6-mercaptopurine (6-MP)
and/or prolonged administrations of asparaginase in consolidation/intensification.
Reinduction treatment mainly consists of a late repetition of the initial remission induction
and early intensification phases. A randomized trial by the Children’s Cancer Group
applying an augmented BFM protocol showed that intensified consolidation and doubledelayed intensification can further improve the outcome of high-risk patients with a slow
initial treatment response. Of interest in this context, in a recent subsequent trial on higherrisk patients with a rapid marrow response to induction therapy by the same group, the
investigators demonstrated an improved event-free survival for more intensive but not for
longer postinduction intensification treatment. Unfortunately, further intensification of
treatment and the related higher doses of glucocorticoids have been associated with a high
incidence of osteonecrosis, especially in older children. Consequently, some investigators
suggest glucocorticoid administration in intensification/consolidation on alternate weeks for
children older than 10 years to improve complication rates.
Central nervous system-directed therapy
CNS-directed therapy has become a prerequisite for successful treatment of childhood ALL.
Before its introduction in the 1960s, more than 50% of children with ALL suffered from
disease recurrence originating from the CNS. This high rate could be reduced to less than
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5% through the introduction of cranial irradiation, intrathecal chemotherapy with
methotrexate alone or in combination with other drugs (cytarabine, hydrocortisone), and
systemic application of chemotherapeutics with adequate penetration into the CNS (highdose methotrexate, dexamethasone, high-dose cytarabine). The intensity of CNS-directed
treatment is adjusted according to the risk of ALL relapse originating from the CNS, the
most important risk factor being overt CNS involvement at diagnosis (CNS3). Additional risk
factors include a high initial white blood cell count, pro-B or precursor T-cell
immunophenotype, t(9;22) or t(4;11), and a traumatic lumbar puncture with identifiable blast
cells present at diagnosis. CNS-directed therapy may differ in the number of intrathecal
injections and/or intrathecally applied drugs, as well as in the inclusion of cranial irradiation
at different doses. Excluding infants, most clinical protocols administering intensive
systemic therapy still recommend preventive cranial irradiation (12 or 18 Gy) for high-risk
patients and/or those with a precursor T-cell immunophenotype - at least for those with
white blood cell counts of 100.000/µl or more at diagnosis. Patients with CNS2 status or a
traumatic lumbar puncture are recommended to receive additional therapeutic doses of
intrathecal chemotherapy. Also CNS3 patients receive more intense intrathecal
chemotherapy and, in addition, are subject to therapeutic cranial irradiation (18 or 24 Gy
when ≥ two years of age; younger children should receive reduced doses). All other patients
(precursor B-cell ALL, CNS1, non HR) should receive preventive intrathecal chemotherapy.
Allogeneic hematopoietic stem cell transplantation
Results of frontline and relapse protocols have improved over time. At the same time, the
experience gained also led to advancements in HSCT procedures. The continuous parallel
developments in both fields complicate the description of the exact role of HSCT in
childhood ALL and elucidate the strong need for prospective clinical trials. Therefore, in
2003, the ALL-BFM and the ALL-REZ BFM study groups initiated a prospective,
international, multicenter trial (ALL-SCT-BFM 2003), which will now be extended to a larger
international consortium. In this trial exactly defined procedures on HLA-typing, donor
selection, conditioning regimen, graft versus host disease prophylaxis and therapy as well
as standards of supportive care ensure a high degree of standardization with regard to all
relevant components potentially associated with the heterogeneity in outcome observed in
the context of HSCT. It is expected that the results of such prospective trials will more
precisely determine the potential of the different HSCT procedures in high-risk or relapsed
childhood ALL. Meanwhile, HSCT in children with ALL in first remission should be confined
to patients whose disease is associated with poor prognostic features such as the t(9;22) or
a poor response to remission induction therapy.
Maintenance therapy
Hypothetically, maintenance treatment aims at a further stabilization of remission by
suppressing the re-emergence of a drug-resistant clone through consistently reducing the
pool of residual leukemic cells. The current standard of maintenance therapy consists of up
to two or three years of treatment (from initial time of diagnosis) with daily oral 6-MP and
weekly oral methotrexate. The combination of 6-MP with methotrexate acts synergistically
as methotrexate inhibits purine de novo synthesis, leading to a higher intracellular Availabi-
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lity and increased incorporation of phosphorylated thiopurines in DNA and RNA. During
maintenance treatment, 6-MP and methotrexate doses are adjusted according to absolute
leukocyte or neutrophil and platelet counts. Important to note and a potential source of
heterogeneity with regard to outcome analyses, the starting dose as well as dose
adjustment guidelines while on therapy may differ between the different study groups. As
several reports suggested an improved outcome with bedtime administration, 6-MP is
commonly administered in the evening hours. Also, 6-MP should not be given in
combination with milk since the xanthine oxidase activity contained in milk decreases the
bioavailability of 6-MP. Of utmost clinical importance, at St. Jude Children’s Research
Hospital researchers have demonstrated that maintaining the highest tolerable dose of daily
6-MP in maintenance therapy is an important prognostic factor in childhood ALL.
Intensification of maintenance treatment by the administration of vincristine/dexamethasone
pulses was recently shown to provide no extra benefit. The reduction of maintenance below
two years (from the time point of initial diagnosis) was associated with an increased
frequency of leukemic relapses. Although it was proven disadvantageous to shorten
maintenance treatment, whether or not extended maintenance of up to three years is
offering any beneficial effect for particular subgroups in the context of different treatment
strategies is not completely evaluated. With regard to the debate on the better thiopurine,
three randomized studies compared the toxicity and efficacy of 6-thioguanine (6-TG) with 6MP in interim maintenance and maintenance therapy of childhood ALL. However, due to the
observation of dose-dependent high rates of severe hepatotoxic side effects associated
with the application of 6-thioguanine, the current thiopurine drug of choice for maintenance
treatment remains 6-MP.
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Table 1. Important prognostic factorsa and their approximate incidences in childhood ALL
Factor
Favorable prognostic factors and their
Unfavorable or less favorable prognostic factors
approximate incidence (%)
and their approximate incidence (%)
Age at diagnosis
≥ 1 and < 10 years (77%)
< 1 year (3%) or ≥ 10 years (20%)
Gender
female (45%)
male (55%)
White blood cell count at diagnosis
Immunophenotype
< 50.000/µl (80%)
CD10-positive precursor B-cell ALL (83%)
CNS diseaseb
CNS 1 (80%)
Genetic featuresc
hyperdiploidy (20%),
TEL/AML1 positivity (20%)
≥ 50.000/µl (20%)
CD10-negative precursor B-cell ALL (4%),
T-ALL (13%)
CNS 3 (3%),
TLP+ (7%)
hypodiploidy (1%),
t(9;22) or BCR/ABL1 positivity (2%),
t(4;11) or MLL/AF4 positivity (2%)
Prednisone responsed
< 1000/µl blood blasts (90%)
≥ 1000/µl blood blasts (10%)
Early bone marrow response
< 5% blasts (M1) on day 15 of induction treatment
(60%)
≥ 25% blasts (M3) on day 15 of induction
treatment (15%)
Remission status after induction therapy in
the bone marrow (morphologically
assessed)
Minimal residual diseasee in the bone
marrow (molecularly assessed )
< 5% blasts (M1) after 4 to 5 weeks of induction
treatment (98%)
≥ 5% blasts (M2 or M3) after 4 to 5 weeks of
induction therapy (2%)
< 10-4 blasts after 5 weeks of induction treatment
(40%)
≥ 10-3 blasts after 12 weeks of treatment (induction
and consolidation) (10%)
a
prognostic factors are treatment dependent and, therefore, the selection presented in the table above cannot be entirely comprehensive; it reflects the current recommendations of AIEOP-BFM
ALL study group and the Biology & Diagnosis Committee of the I-BFM Study Group .
CNS1 (puncture nontraumatic, no leukemic blasts in the cerebrospinal fluid (CSF) after cytocentrifugation); CNS3 (puncture nontraumatic, >5 leukocytes/µL CSF with identifiable blasts); TLP+
(traumatic lumbar puncture with identifiable leukemic blasts); a TLP with no identifiable blasts is not an adverse factor; the prognostic impact of CNS2 status (puncture nontraumatic, ≤5
leukocytes/µL CSF with identifiable blasts) is debated. For cytomorphological examination, CSF samples should be analyzed after cytospin preparation, a method through which cellular components
within the CSF are concentrated by centrifugation.
c
hyperdiploidy defined as the presence of more than 50 chromosomes or a DNA index (the ratio of DNA content in leukemic G0/G1 cells to that of normal diploid lymphocytes) ≥1.16; hypodiploidy
defined by <45 chromosomes; the prognostic value of MLL gene rearrangements other than MLL/AF4 and presence of the E2A/PBX1 fusion transcript are debated.
d
after 7 days induction with daily prednisone and a single intrathecal dose of methotrexate on treatment day 1.
e
-4
assessed by molecular genetic techniques or flow cytometry; markers required to have a sensitivity of at least 10 .
b
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Diagnosis of childhood acute lymphoblastic leukemia
The diagnosis of ALL should be made, or reviewed, in a laboratory providing the necessary
expertise and adequate technical equipment. A diagnostic setting in a clinical trial
environment on acute leukemias requires access to different resources which can be
centralized in a single place or be present in a decentralized structure in form of a
laboratory network structure.
Diagnostic methods
The diagnostic work-up of childhood ALL requires cytomorphology with cytochemistry,
immunophenotyping, karyotyping, FISH, and molecular genetic analyses of bone marrow
and/or peripheral blood leukemic cells. To tailor the intensity of CNS-directed treatment,
appropriate assessment of CNS involvement needs to be performed. The definition of CNS
disease is explained in Table 1.
Cytomorphology
The morphologic classification of ALL should be based on the French-American-British
(FAB) classification. Cytochemistry may help to assess lineage affiliation (e.g.,
myeloperoxidase [MPO]-positivity). In the presence of ambiguous morphology and
cytochemistry, immunophenotyping should be employed to define lineage. Reference
cytomorphology in the context of clinical trials may increase the quality of cytomorphological
assessments.
Immunophenotyping
In the context of ALL, international efforts within the I-BFM Study Group have led to a
consensus panel to be assessed for all ALL cases which should include iCD3, iCD22,
iCD79a, iIgM, iLysozyme, iMPO as intracellular markers (prefix “i” stands for intracellular
staining; in combination with CD45) as well as CD2 (PE recommended), CD3, CD5, CD7;
CD10, CD19, CD20; CD11b, CD11c, CD13, CD14, CD33, CD64, CD65 (available only in
FITC), CD117; CD34, CD56, and HLA-DR as surface markers (in combination with CD45).
In an integrated approach, the respective lineage-associated MRD-tubes should then be
added according to the primary lineage and, in addition, markers for lineage subclassification be added as appropriate (e.g., if T-ALL: CD1a, TCRab, TCRgd, CD4, CD8; if
B-ALL: CD15; if B-IV suspected: Kappa and Lambda (on surface after pre-washing or
intracellular); if biphenotypic acute leukemia (BAL) according to general panel: CD24, iTdT).
In case of double-platform approaches, screening tubes must include in all cases the
following markers: CD19, CD10, iCD79a, iCD22, iCD3, CD7, iMPO, and CD45.
Permeabilization reagents for intracellular stainings should be applied in a standardized
fashion in clinical trial groups. Bone marrow is preferred over peripheral blood for analyses.
The latter may be used for assessing the immunophenotype if bone marrow is not available
or of poor quantity/quality. There is consensus that lysis preparation is preferred. Additional
usage of SYTO-16 or -41 is recommended to distinguish non-nucleated events
(erythrocytes) from blasts which is particularly relevant in cases of very low CD45
expression of blasts. Immunological blast gating should be performed based on a
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CD45/SSC backbone strategy and lineage-defined gating wherever appropriate. The
percentage of blasts among total nucleated cells of the sample must be recorded. Notably,
formal diagnosis of leukemia and treatment indication usually depends on morphologic blast
enumeration respecting conventional thresholds (e.g. ≥25% as per AIEOP-BFM ALL 2009
protocol, or as per current WHO definition which respects a usual threshold of 20%). This
threshold requirement is, however, independent of the process of immunophenotyping of
the leukemia. Hence, there is no formal cut-off of blast percentage below which the
immunophenotype may not be determined – the leukemic immunophenotype can be
determined as long as the blast population can be clearly distinguished from normal
background cells. In such case with a malignant blast percentage <25% by flow cytometry,
however, the immunophenotypic report should not anticipate formal diagnosis of ALL but
state e.g. “malignant lymphoblasts suggestive of ALL with low percentage of blasts”. In
leukemia with maturing cells in addition to immature blasts (usually not an issue in ALL –
not relating to B-I to B-III transition; but common in AML with maturation, e.g., FAB M2 or
M4), detailed immunophenotype reporting relates only to the population of immature blasts
(most frequently defined by CD34 and/or CD117 expression), but the phenotypic
peculiarities of the maturing leukemia cells (as far as unambiguously belonging to the
leukemia) should be reported in the descriptive summary (including % among all nucleated
cells). In case of truly bilineal leukemia, the detailed phenotype should be reported for the
larger clone, and the details of the second blast population should be summarized in the
descriptive summary (including % among all nucleated cells, lineage and subtype
assignment, antigen expression pattern as different from the larger clone). A minimum of 30
000 nucleated events should be acquired per tube. The definition of antigen distribution and
expression levels should employ ratings which use assessment of antigen expression on
blasts relative to an appropriate negative population. By determining the degree of overlap
(prevalence) of the blast population with the negative control subset, expression is to be
rated as either negative (overlap in ≥90%, i.e. non-overlap in <10% of blasts), weak positive
(non-overlap=positivity in ≥10% to <50% of blasts, i.e. low expression with a major overlap
with negative cells or positive in only a minor separate subset of blasts), or strong positive
(non-overlap=positivity in ≥50% of blasts, i.e. all kinds of expression with non-overlap of the
majority of blasts). In addition to that a more detailed further description of antigen
distribution in combination with expression intensity relative to the appropriate control
population is a recommended optional output. This fine-rating should be employed
according to adapted Bethesda criteria which make use of a descriptive and semiquantitative scale rather than an exact enumeration procedure. Hence, the focus in
interpretation of phenotyping data on the descriptive scale lies more on robustness in daily
routine and less on biologic accuracy. Among “weak positive” distinguish dim positive from
partially positive in <50% of blasts (= partially positive “1”). Among “strong positive”
distinguish medium, bright, heterogeneous or partially positive in ≥50% of blasts (= partially
positive “2”).
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Flow cytometry for determination of DNA index
Determination of DNA index by flow cytometry is frequently employed to indicate the
presence of hidden high hyperdiploid clones (not detected by cytogenetics). These poor risk
abnormalities are often accompanied by a population comprising a doubling of the
hypodiploid clone, which may represent the predominant population. In near-haploid cases
the doubled population may be misclassified as classical high hyperdiploidy. As these two
abnormalities represent poor and good risk groups, respectively, their accurate distinction is
vital and the technique recommended to be employed in the diagnostic wotk-up of clinical
trials on treatment of childhood ALL.
Cytogenetic analysis of bone marrow blasts allows for the identification of significant
numerical and structural chromosomal abnormalities in acute leukemias. In childhood ALL,
the poor risk group currently comprises patients with t(9;22)(q32;q11), translocations
involving the MLL gene, primarily t(4;11)(q21;q23), near-haploidy and iAMP21. FISH is one
of the most successful techniques to be integrated into routine clinical practice recently. It
offers a valuable complementary approach to cytogenetics and one main advantage is that
it provides information in addition to the result of the specific test for which it is applied. For
diagnostic purposes, FISH should always be carried out in accredited laboratories with a
high throughput of samples. Preparation and analysis should always be performed in
accordance with national quality assessment schemes. In diagnostic assessment
procedures, highest priority should be given to the high-risk abnormalities, in particular:
BCR-ABL1; MLL rearrangements, particularly t(4;11)(q21;q23) giving rise to the MLL-AFF1
fusion; iAMP21; and near-haploidy. The good risk abnormalities, ETV6-RUNX1 fusion and
hidden high hyperdiploidy, are also recommended to be assessed with high priority for
testing as they are cryptic at the cytogenetic level. In addition, their presence does not only
impact on patient management but the same probes are used for the detection of the poor
risk anomalies: iAMP21 and near-haploidy.
Molecular genetics
Molecular genetic techniques have revolutionized leukemia diagnostics and have the
advantage of making use of DNA or RNA extracted directly from bone marrow without the
need for cell culture to produce metaphases or laborious chromosomal analysis. With
increasing numbers of significant chromosomal translocations and their partner genes
identified, techniques of reverse transcriptase PCR (RT-PCR) were developed for their
detection. The advantage of this approach is that it requires only small amounts of RNA to
demonstrate the expression of the fusion transcript arising from the significant
translocations. RT-PCR is used as a complementary or alternative technique to cytogenetic
analysis in a number of protocols for the rapid detection of specific translocations. It
provides a highly sensitive and accurate method, even in a multiplex reaction, for the
identification of specified abnormalities among patients with a failed cytogenetic result or
variant and complex chromosomal rearrangements. One major disadvantage is that
cooperating abnormalities cannot be simultaneously identified. Recently, Multiplex Ligation-
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dependent Probe Amplification (MLPA) became an important technique in the molecular
work-up of specific losses and gains of genetic material in acute leukemias.
Due to the integrated use of cytogenetic and molecular genetic techniques in a
complementary way to assess the most relevant recurrent genetic aberrations, both
technical approaches are described below in context of those recurrent aberrations
recommended to be assessed.
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BCR-ABL1 fusion
Routine cytogenetic analysis and RT-PCR readily detects the t(9;22)(q34;q11)/BCR-ABL1
fusion in the majority of patients. Those with a normal or “ill-defined” karyotype, a failed
cytogenetic result or a discrepancy between the cytogenetic and molecular result should be
tested by FISH. The use of a dual colour, dual fusion probe is recommended. The detection
of two fusion signals indicates a positive result with both fusion products present, for which
the false positive rate is extremely low. At the same time these probes will show the
presence of a second copy of the Philadelphia (Ph) chromosome, which is a characteristic
feature of Ph-positive ALL and indicate deletions from the derivative chromosome 9
involving the reciprocal ABL1-BCR fusion. These probes will also detect the NUP214-ABL1
fusion, found as a secondary change in T-ALL and other rarely occurring ABL1
translocations, which may be responsive to imatinib treatment. It could be argued that the
presence of the BCR-ABL1 fusion is the significant event; therefore FISH or RT-PCR could
replace cytogenetic analysis for the detection of this abnormality. However, for the purpose
of monitoring patients following treatment with imatinib, it is important to be aware of
secondary or chromosomal changes acquired during therapy.
MLL fusion
For risk stratification in current childhood ALL treatment trials, the translocation
t(4;11)(q21;q23)/MLL-AFF1 fusion is classified as high-risk, although the prognosis of the
other MLL partners may become significant in the future. As for BCR-ABL1, the
t(4;11)(q21;q23)/MLL-AFF1 fusion is readily detectable by cytogenetics and RT-PCR.
Multiplex molecular approaches may be applied for the detection of this fusion in
conjunction with other common MLL translocations. A dual colour breakapart probe
provides a highly effective approach for the detection of all chromosomal rearrangements
involving MLL by FISH, including variant t(4;11) translocations and cryptic insertions. This
FISH probe also detects 3’ deletions associated with MLL translocations. This confirms, that
as for BCR-ABL1, those samples with a normal or “ill-defined” karyotype, a failed
cytogenetic result or a discrepancy between the cytogenetic and molecular result should be
tested by FISH.
ETV6-RUNX1 fusion
This fusion arises from the translocation t(12;21)(p13;q22). Although the fusion transcript
can be detected by RT-PCR, the translocation is kryptic at the cytogenetic level. An extra
signal or dual colour fusion probe is usually used for detection by FISH. The advantage of
the FISH approach is that fusions arising from rare variant breakpoints are also identified,
which may not be detected by RT-PCR. In ETV6-RUNX1 positive cases, FISH provides
information on the status of the second ETV6 homologue: whether it is retained or deleted,
as well as indicating the number of RUNX1 and fusion signals. Deletion of the second ETV6
allele and additional fusion signals are important secondary events in this ALL subtype,
which may be linkable to outcome in future stuies and, therefore, are recommended to be
assessed.
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Hidden high hyperdiploidy
In ETV6-RUNX1-negative cases, additional RUNX1 signals are frequently observed in
interphase. In high hyperdiploid cases, these usually correspond to the additional copies of
chromosome 21 seen by cytogenetic analysis, which are a characteristic finding in this
cytogenetic subgroup. Thus it can be inferred that additional RUNX1 signals seen in
interphase cells of patients with normal of failed cytogenetic results, when negative for the
three abnormalities: ETV6-RUNX1, BCR-ABL1 fusions and MLL rearrangements, represent
additional copies of chromosomes 21, likely within a hidden high hyperdiploid clone. When
using the ETV6-RUNX1 probe, the ETV6 signals will indicate the number of copies of
chromosome 12, acting as an internal control for RUNX1. Chromosomes 9, 11 and 22 are
less frequently gained than chromosome 21 within high hyperdiploid karyotypes, therefore
the relative numbers of signals seen with the BCR-ABL1 and MLL probes will rule out the
presence of triploid or tetraploid populations. Subsequent FISH hybridisation with
centromeric probes specific for the simultaneous trisomies of chromosomes 4, 10, and 17 is
recommended to identify the good risk group of high hyperdiploid patients as defined by the
Children’s Oncology Group.
iAMP21
iAMP21-positive samples are negative for the fusion, while in addition to the two normal
copies of the ETV6 signal, show multiple RUNX1 signals (3 or more additional signals) with
the ETV6-RUNX1 probe. In metaphase, one signal is located to the normal chromosome
21, while the others are seen in tandem duplication along an abnormal chromosome 21. In
interphase, the signals are clustered together, except for one signal representing the normal
chromosome 21 which is usually located apart. The observation that the commonly
amplified region always includes the RUNX1 gene, confirms that probes directed to RUNX1
provide a reliable detection method for this abnormality. It was shown by FISH, using a
probe specific to the 21q subtelomeric region, and aCGH that the abnormal chromosome
21 frequently shows a deletion of the subtelomeric region. Alternatively a normal pattern is
seen. In rare cases where a 21q subtelomere is gained, the level of RUNX1 amplification is
always greater. This means that accurate distinction of iAMP21 from the gains of whole
chromosomes 21 or isochromosomes 21 in interphase can be made by calculating the ratio
of RUNX1 to subtelomeric signals. When whole chromosomes 21 are gained or an
isochromosome 21 is present, the number of RUNX1 and 21q subtelomeric signals will be
equal in number, with a ratio of 1. In iAMP21 the number of RUNX1 signals will always
exceed the number of signals from 21q, with a ratio greater than 1. Thus application of
these two probes directed to chromosome 21 provides an accurate interphase FISH
diagnostic test for iAMP21.
Hidden near haploidy
Although near-haploidy is classified as a high risk abnormality in childhood ALL, it is rare.
Its characteristic features are the gain of specific chromosomes onto the haploid
chromosome set and, in the majority of patients, the presence of a population of cells with
an exact doubling of this chromosome number. This doubled population is often the
predominant one. The majority of near-haploid cases are identified from cytogenetic analy17
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sis or their presence is inferred by the finding of the doubled population. This mixture of
near-haploid and doubled populations makes FISH detection of hidden populations difficult.
Probes specific for chromosomes which would be expected to be present as a single copy
in the near-haploid clone need to be applied in parallel with probes specific for
chromosomes which would be expected to have four copies within the doubled population.
As chromosomes 3, 7, 9, 11 and 22 are rarely gained in near-haploidy, centromeric probes
to chromosomes 3 and 7, in addition to the BCR-ABL1 and MLL probes should identify the
near-haploid clone. In these cases flow cytometry to measure the DNA index of the two
populations may provide a more appropriate and accurate detection method.
IGH@ translocations
Translocations involving IGH@ at 14q32 are emerging as a significant subgroup in
childhood ALL. Dual colour breakapart probes can identify these translocations and in
metaphase will identify their partners allowing studies to inform of their biological and
prognostic significance. This probe will also indicate gains of chromosome 14 in hidden high
hyperdiploid cases.
t(17;19)(q22;p13)
The translocation, t(1;19)(q23;p13)/TCF3-PBX1, fusion is now regarded as a standard risk
abnormality in childhood BCP-ALL. It has been shown that not all translocations involving
t(1;19)(q23;p13) at the cytogenetic level involve this fusion. This has been demonstrated by
FISH using a breakapart probe to TCF3. In addition this probe is able to detect the
presence of variant translocations involving TCF3. Of note is the t(17;19)(q22;p13) with the
TCF3-HLF fusion, which even on current therapies has a very dismal outcome. Although
this translocation is rare, it is usually visible by cytogenetic analysis and primers exist for its
detection by RT-PCR. However, in cases with failed and normal cytogenetic results FISH
with the TCF3 probe would be valuable to ensure that all cases are identified in view of the
high risk associated with this abnormality.
CDKN2A deletions
FISH with a commercially available probe to CDKN2A provides a reliable method for the
detection of deletions around this gene. Although this is known to be an important
secondary abnormality in both BCP- and T-ALL, its relative incidence among the different
cytogenetic subgroups and its relative prognostic significance remain unclear. Routine FISH
screening within different treatment protocols will be of value to inform the design of future
treatment protocols.
IKZF1 and CRLF2
As further evidence is accumulating regarding their potential inclusion in future riskstratification algorithms, deletions of IKZF1 and the PAR region on chromosome X including
CRLF2 is recommended to be assessed by MLPA. In addition, other techniques for the
assessment of aberrant IKZF1 and/or CRLF2 (e.g., real-time quantitative PCR, fusion gene
expression bei RT-PCR, and cell surface expression by flow cytometry are strongly encou-
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raged to be evaluated in clinical trials on ALL as their exact prognostic value is yet to be
defined.
TPMT
To prevent prolonged hematopoietic toxicity for TPMT deficient individuals upon exposure
towards thiopurine drugs (6-MP, 6-TG), all patients with ALL should be genotyped for the
most common variant TPMT alleles before introduction of thiopurine medications (TPMT*2,
and *3A, *3C).
Response to treatment is the most important indicator of outcome in childhood ALL. Most
study groups evaluate treatment response by cytomorphology in the bone marrow after
treatment days 8, 14, or 15, and after the end of induction therapy (e.g., days 28 or 33).
Measures of cytomorphological treatment response are relevant for stratification on many
protocols. The difficulties of assessing hypoplastic bone marrow are well acknowledged
here. Besides cytomorphological response assessment, MRD analyses by mainly two
technical approaches, DNA-based PCR of clone-specific Ig or TCR gene rearrangements or
assessment of an aberrant immunophenotype by flow cytometry (see also section on
immunophenotyping), have become the main stratification elements in childhood ALL. It has
to be stressed that MRD analyses should be incorporated in the context of clinical trials
and, if appropriate, for treatment stratification. However, the latter can only be
recommended in association with the existing international study groups on MRD to assure
comparable and quality-controlled procedures.
Biobanking
Biobanking of spare diagnostic material in the context of clinical trials according to
standardized operating procedures is recommended. Consent issues should be addressed
already in the trial protocols.
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Recommendations for the diagnosis of childhood acute myeloid
leukemia (AML)
General information
Childhood AML is rarer than ALL and accounts for up to 20% of childhood leukemias. The
annual incidence rate of this disease varies around a half new case per 100,000 children
younger than 15 years. Mainly through joint efforts of national cooperative trial groups,
survival rates during the last decades increased to 70%. These results are reached by
application of intensive multiagent chemotherapeutic regimens and in specific patient
subgroups additional radiotherapy and/or hematopoietic stem cell transplantation (HSCT).
Treatment regimens basically consist of three main components: (1) an induction period; (2)
consolidation/intensification elements; and (3) central nervous system (CNS) preventive
therapy. Treatment duration is approximately 18 months. Certain clinically and biologically
distinct patient subgroups with AML receive treatment specifically adapted to their needs
(e.g., ATRA-based maintenance treatment in promeyelocytic leukemia, adapted
chemotherapeutic protocols for patients with Down syndrom).
Similar to ALL, the initial clinical presentation of a child with AML largely depends on the
extent of the leukemic infiltration of the bone marrow and extramedullary sites. The
diagnosis of ALL is usually made by cytomorphological and cytochemical examination of a
bone marrow aspirate. CNS involvement (CNS3 status) is diagnosed by the presence of
blasts in the cerebrospinal fluid (CSF) or if intracerebral infiltrates are detected by crosssectional radiological imaging. Initial diagnostics are complemented by flowcytometry-based
immunophenotyping to gain information on the blasts expression of myeloid differentiationassociated antigens as measured by the reactivity to specific monoclonal antibodies. In
addition, a combined approach using cytogenetic and molecular genetic techniques is used
for the detection of genetic aberrations, such as non-random recurrent structural and, less
frequently, numerical chromosomal aberrations (e.g., different MLL rearrangements, t(8;21),
inv(16), t(15;17), t(7;12), t(6;9), monosomy 7). Molecular-genetic techniques are employed
to detect additional somatic aberrations (e.g.; FLT3 internal tandem duplications (ITD),
mutations of WT1, N-RAS, K-RAS, PTPN11, and c-KIT).
Large research efforts on the clinical and biological aspects of AML have led to the
identification of several prognostic factors – mostly genetic aberrations – which are used for
risk-adapted therapeutic treatment stratification in the clinical management of childhood
AML. Response to the first course of treatment and genetics are the most important
prognostic factors and are instrumental for most of today’s risk group stratification
strategies. Most study groups evaluate treatment response morphologically in the bone
marrow after the first and second induction course (e.g.; treatment days 15 and 28). MRD in
AML can be analyzed by morphology, immunophenotyping, and quantification of molecular
aberrations as well as specific gene expression. The definite clinical implications of the
different MRD measurements are still under debate.
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Remission induction treatment
Contemporary treatment approaches for childhood AML mostly apply two courses of
induction therapy which routinely consist of three drugs – an anthracycline (eg.;
daunorubicin or idarubicin), cytarabine, and etoposide or 6-TG – and lead to achievement of
complete remission in more than 85% of patients.
Consolidation/intensification treatment
Eradication of residual leukemic blasts in patients who are in remission by morphologic
criteria is the primary aim of consolidation/intensification treatment. In childhood AML this is
achieved by application of chemotherapy courses consisting of drug combinations similar to
those given during induction. The value of applying more than three consolidation courses
is unclear.
Central nervous system-directed therapy
CNS-directed therapy has become a prerequisite for successful treatment of childhood
AML. Similar to ALL, preventive CNS treatment is given to all patients and traditionally
consisted of intrathecal chemotherapy (cytarabine or methotrexate, or triple drug
combinations of cytarabine, methotrexate, and hydrocortisone). Depending on the
protocols, intrathecal chemotherapy was given alone or combined with cranial irradiation.
During the last decade, however, systemic chemotherapy regimens with adequate CNS
intensity together with the lack of evidence of superiority for cranial irradiation led to
abandonment of irradiation by all major study groups. A CNS3 status in AML is not a crucial
factor within the AML risk group stratification because it does not affect overall survival.
However, those with CNS involvement relapse more frequently in the CNS and, therefore,
require intensified intrathecal therapy. Although most study groups have added CNS
irradiation to the regimen of these patients, recent observations suggest that frequent
intrathecal chemotherapy combined with intensive systemic chemotherapy may yield similar
results.
Allogeneic hematopoietic stem cell transplantation
The role of allogeneic HSCT in first complete remission is not clearly defined, but HSCT in
second CR is generally considered.
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Diagnosis of childhood acute myeloid leukemia
The diagnosis of AML should be made, or reviewed, in a laboratory providing the necessary
expertise and adequate technical equipment. A diagnostic setting in a clinical trial
environment on acute leukemias requires access to different resources which can be
centralized in a single place or be present in a decentralized structure in form of a
laboratory network structure.
Diagnostic methods
The diagnostic work-up of childhood AML requires cytomorphology with cytochemistry,
immunophenotyping, karyotyping, FISH, and molecular genetic analyses of bone marrow
and/or peripheral blood leukemic cells. To tailor the intensity of CNS-directed treatment,
appropriate assessment of CNS involvement needs to be performed. The definition of CNS
disease is explained in Table 1.
Cytomorphology
AML classification is based on lineage-associated phenotype (undifferentiated, myeloid,
monoblastic, erythroblastic, or megakaryoblastic) and defined according to FAB.
Cytochemistry confirms lineage affiliation and classifies myeloid (MPO-positivity) and
monoblastic differentiation (nonspecific esterase-positivity). In the presence of ambiguous
morphology and cytochemistry, immunophenotyping may further support the lineage
definition. Acute megakaryoblastic leukemia (AMKL, FAB M7) and minimally differentiated
AML (FAB M0) have to be confirmed by immunophenotyping, although the former may
show typical morphologic features. The presence of myelofibrosis frequently associated
with acute megakaryoblastic leukemia, and consequent sampling problems may lead to an
underestimation of blasts by both morphology and immunophenotyping. In case of a low
blast count (< 20%), repeated bone marrow sampling, including biopsy, needs to be
performed.
Immunophenotyping
At present, there is no standardization of antibody panels used for immunophenotyping
among the large trial groups. However, upcoming standards suggest the use of multicolor
monoclonal antibody combinations that include CD45 to enable optimal gating and analysis
of the blast population within the complex context of residual hematopoiesis. In addition,
these procedures might improve flow cytometry techniques for assessing MRD in childhood
AML. Another methodologic transition concerns the interpretation of immunophenotypic
expression data. Rigid cut-off points for determining marker positivity (e.g., 10% or 20%)
are being replaced by biologically more meaningful semiquantitative estimates of blast
population appearances compared with those of normal populations. The mandatory
minimal panel required to fulfill WHO and EGIL criteria for AML includes CD34, CD117,
CD11b, CD11c, CD13, CD14, CD15, CD33, CD64, CD65, iMPO, i-lysozyme, CD41, and
CD61; and for MPAL: CD19, iCD79a, iCD22, CD10, and iCD3.
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Cytogenetic analysis is required to allow detection of specific recurrent structural and
numerical cytogenetic abnormalities present in childhood AML. The most frequent
chromosomal abnormalities in children with AML include t(8;21)(q22;q22),
inv(16)(p13.1q22)
(together
referred
as
core
binding
factor
[CBF]-AML),
t(15;17)(q22;q21)/PML-RARA, and 11q23/MLL-rearranged abnormalities (up to 25%),
which together account for approximately 50% of childhood AML cases. Importantly, the
2008 WHO classification categorizes t(9;11)(p22;q23)/MLL-MLLT3(AF9) as an entity and
recommends that partners in the variant MLL translocations should be identified.
Frequently, 11q23/MLL rearrangements are complex and/or cryptic. For example, the
t(10;11)(p12;q23)/MLL-MLLT10(AF10) or t(6;11)(q27;q23)/MLL-MLLT4(AF6) can generate
the fusion via inversions, insertions, or translocations involving other chromosomes. The
best method to evaluate these cases is the sequential G-banding to FISH with a MLL probe
or RT-PCR, using specific primers. Here, cytogenetics have to be part of an integrated
diagnostic apprach. Monosomy 7, monosomy 5/5q deletions, and aberrations of 12p are
rare events (seen in 3%-5% of patients) that occur in nearly all subtypes of childhood AML.
Trisomies 8 und 21 are often associated with additional aberrations. Overall, routine
evaluation should include the evaluation of prognostically relevant genetic aberrations by
cytogenetics/FISH, including at least the following fusion genes at diagnosis: RUNX1RUNX1T1, CBFB-MYH11, PML-RARA, and MLL rearrangements. Other rare fusion genes
should be traced to determine adverse risk patients in the context of clinical trials.
Molecular genetics
Several gene mutations and aberrantly expressed genes have been described in childhood
AML. In AML with a normal karyotype (CN-AML), several mutations, such as NPM1, FLT3,
WT1, and biallelic CEPBA mutations, are clinically relevant and should be included in
standard diagnostics. Mutations in the WT1 gene are found mainly in CN-AML and are
often associated with FLT3-ITD mutations. The frequency of activating mutations of tyrosine
kinase receptor genes, such as FLT3 (predominantly in CN-AML, t(15;17)(q22;q21)/PMLRARA and t(5;11)(q35;p15.5)/NUP98-NSD1), increases with age. Point mutations in the
activating loop domain of the FLT3 receptor (frequency 2%-8%) are mutually exclusive of
FLT3-ITD mutations. Mutations in genes involved in the RAS-RAF-ERK signal transduction
pathway (PTPN11, NF-1, N-RAS, K-RAS) occur in 5%-21% of children with AML, more
frequently in those with core binding factor (CBF)-AML, and in young children with MLLrearranged AML. C-KIT mutations occur in approximately 25% of children with CBF-AML,
but in only 5%-8% of those with other leukemia types. Furthermore, it is regarded desirable
to assess prognostically relevant MLL fusions genes. At a minimum consesus, routine
evaluation should include the evaluation of a prognostically relevant and potentially
targetably selected set of molecular genetic markers FLT3-ITD, WT1, C-KIT, CEBPA
(double mutation), NPM1, and further specific MLL-abnormalities with favorable or very
poor prognosis (e.g., MLL-AF1Q, AF6, AF10).
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Response to the first course of treatment is an important indicator of outcome. Most study
groups evaluate treatment response by cytomorphology in the bone marrow after the first
(e.g., on treatment days 15 or 28) and second induction courses and employ the
information for risk group stratification. The difficulties of assessing hypoplastic bone
marrow are well acknowledged here. Although the overall definitve prognostic value of MRD
analyses in AML still under debate, MRD monitoring can be applied in subsets of childhood
AML by employing different technical approaches. MRD assessment by
immunophenotyping can be done in up to 96% of children with AML. However, the
heterogeneity of leukemia-associated immunophenotypes and frequent antigen shifts over
time can limit the sensitivity and specificity of immunophenotypic detection of MRD. Recent
technological advances, such as multi-color flow cytometry, may overcome these
limitations. However, standardization of methods and quality control measures are of
utmost importance to assure comparability of data. At the transcript level, the high
specificity and sensitivity of real-time quantitative PCR for the detection of AML fusion
genes (e.g., RUNX1(AML1)-RUNX1T1(ETO), CBFB-MYH11, PML-RARA, and
MLLT3(AF9)-MLL) represents a monitoring option for aaproximately one third of patients.
After evident assessment of their clinical value, these measurements may provide guidance
for decision making on early HSCT or re-introduction of chemotherapy. Another option for
MRD monitoring employs monitoring specific mutations as a clone-specific marker (e.g.,
NPM1, FLT3-ITD, or GATA1s). Overall, MRD measurement should be incorporated in the
context of clinical trials and used, if appropriate, for treatment stratification.
Biobanking
Biobanking of spare diagnostic material in the context of clinical trials according to
standardized operating procedures is recommended. Consent issues should be addressed
already in the trial protocols.
24
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35

ENCCA

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