Accuracy Testing of the Chromosomal Aberration and Gene Mutation Markers of the AMLProfiler

Acute myeloid leukemia is a bone marrow malignancy of progenitor cells of the
myeloid cell lineage it includes APL (Acute Promyelocytic Leukemia) and MDS
which resembles AML, and is the most common type of leukemia in adults. There
are multiple varieties of acute myeloid leukemia with characteristic prognosis.
Estimated new cases from acute myeloid leukemia (AML) in the western world in
2009 amount to 31.500 cases annually.

The World Health Organization (WHO) classification of AML incorporates and
interrelates morphology, cytogenetics, molecular genetics, and immunologic
markers as a classification that is universally applicable and prognostically
valid. An important part of this classification is based on genetic analysis of
the blast cells from the patients. Amongst others, CBFB-MYH11 fusion, AML1-ETO
fusion, PML-RARA fusion, NPM1 mutations, and CEBPA mutations are frequent and
clinically relevant genetic aberrations in AML and APL .

Molecular classification of AML, APL and MDS patients is performed using gene
profiles of bone marrow aspirates collected at the time of diagnosis. Three
markers for chromosomal aberrations, one for the detection of CEBPA double
mutants, and one for NPM1 mutation detection are included in the AMLProfiler.
Collectively these markers allow the identification of inv(16) / t(16;16),
t(15;17), t(8;21), NPM1 mutant and CEBPA double mutant cases(see table 2). The
incidence of these aberrations and mutations in the patient population are
summarized below.

Incidence rate of genetic aberrations involved in AML and APL
t(8;21): 8 % Prevalentie
t(15;17): 5 - 10 % Prevalentie
inv(16)/t(16;16): 5 - 10 % Prevalentie
NPM1 mutanten (A, B or D): 25 - 40 % Prevalentie
CEBPA dubbel mutanten: 4 - 5 % Prevalentie


Intended use of the AMLprofiler assay:
The AMLProfiler assay is a qualitative in vitro diagnostic test for the
detection of AML,APL and RAEB specific chromosomal aberrations (specific
recurrent translocations and inversions), as well as expression of specific
genetic markers in RNA extracted from bone marrow aspirates of patients with
Acute Myeloid Leukemia. The results of the AMLProfiler assay may be used to aid
in the diagnosis or the assessment of prognosis of AML, APL and RAEB.

Markers associated with the AMLProfiler are:
Chromosomal abberations: t(8;21), t(15;17) en inv(16) / t(16;16)
Mutations: CEBPA gen (dubbel mutaties), NPM1 gen
Gene expression: BAALC, EVI1

The AMLProfiler is intended for professional use only. For in vitro diagnostic
use.

The objective of the study is to demonstrate the accuracy (positive and
negative percent agreement) by comparing AMLProfiler results from fresh and
banked, AML, APL and RAEB bone marrow samples at multiple participating
clinical sites with results from the sequencing reference lab.
The AMLProfiler molecular diagnostic assay also contains expression markers in
the array that will be evaluated in the protocol Prognostic value of the
Expression Markers of the AMLProfiler, and are not covered in this study.

The design is a comparison study in which AMLProfiler results for the markers
t(8;21), t(15;17), inv(16) / t(16;16), CEBPA double mutants and NPM1 mutation
will be compared to those obtained using standard sequencing methods generated
at the sequencing reference lab.
The positive and negative percent agreement of the AMLProfiler to the molecular
result will be calculated using results generated from 109 positive samples for
each marker derived from 200 prospective (fresh), uncharacterized bone marrow
samples, from 4 participating clinical sites in the EU (3) and US (1) with
additional banked samples (total of 545 samples).

Problems associated with diagnostic bone marrow aspiration are highly uncommon.
Bone marrow punctures may be associated with local discomfort or pain at site
of aspiration and infrequently with:
• Bleeding at the sample collection site.
• Local infection at the sample collection site.
There are no potential benefits to the subject