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Guide And Atlas For The Detection Of Minimal Residual Disease In AML

The past decade has brought new technologies to the study of minimal residual disease (MRD) in acute myelogenous leukemia (AML), which have contributed greatly to the understanding of the term "remission" in this disease. Determination of MRD is an evolving field in which the technology and understanding of the results are continually being refined. Pathologic examination, cytogenetics, fluorescence-activated cell-sorting (FACS) analysis, and now polymerase chain reaction (PCR) all detect, albeit with differing sensitivities, MRD. It is hoped that the clinician and scientist will be able to use these and tomorrow's tools in order to advance clinical management. The following shows various techniques for measuring MRD and their detection limits.[1]

Pathologic Examination is still regarded as the standard but is the least sensitive technique (detection limits 10-1 to 10-2).

Technique

Marker

Detection Limits

Routine pathology

Cellular morphology

10-1 to 10-2

Cytogenetic Analysis has a sensitivity comparable to that of pathologic evaluation; however, this is useful for only the approximate 60% of patients who have an abnormality detected in the original blast population.[2,3]

The table below outlines some of the more common cytogenetic abnormalities and their associated molecular changes.[4]

Technique

Marker

Detection Limits

Cytogenetics

Chromosome morphology

10-1 to 10-2

Common Structural Cytogenetic Abnormalities and Associated Molecular Changes in AML[4]

FAB Class

Cytogenetic Changes

Molecular Changes

M1

t(9;22)(q34;q11)*

BCR/ABL

M2

t(8;21)(q22;q22)
t(6;9)(p23;q34)**

AML1/ETO
DEK/CAN

M3

t(15;17)(q22;q11)¹

PML/RAR-alpha

M4

inv(16)(p13;q22)
t(16;16)(p13;q22)§

CBFß
CBFß/MYH11

M5

t(11;V)II(q23;V)II

MLL/variousII

M6

inv(3)(q21;q26)¶
t(3;3)(q21;q26)¶

MDS1
(EVI-1;EAP)

M7

t(3;21)(q26;q22)#

MDS1(EVI-1;
EAP)/AML1

AML=acute myelogenous leukemia.
*Also occurs in M2; only detected in 2% of AML patients.
** May be seen in M4 and other types of AML; often associated with marrow basophilia.
¹ Pathognomonic of APL.
§ Pathognomonic of AML-M4EO (often associated with marrow eosinophilia).
II

1q21

AF1q

9p22

AF9

19p13.1

ELL/MEN

1p32

AF1p

10q12

AF10

22q11

AF22

4q21

FEL/AF4

17q21

AF17

Xp13

AFX1

6q27

AF6

19p13.3

ENL

¶ Changes affecting 3q26 may be seen in M1, M4, and M7.
# May also be seen in Ph+CML in crisis and therapy-related MDS.

Florescence In Situ Hybridization (FISH) uses specific probes that detect the presence or absence of particular chromosomes (centromeric probes) or abnormal sequences (cosmid probes). For example, FISH can be used to detect trisomy 8(+8) or deletion of chromosome 7(del 7); these are common abnormalities in older patients with AML or in patients with secondary leukemia.[3] While the sensitivity is similar to that of routine cytogenetics, one advantage of this technique is that dividing cells are not required. The examination can be performed on interphase nuclei, and a large number of cells can be analyzed in a short period of time.

Technique

Marker

Detection
Limits

FISH

Chromosome structure

10-2

Florescence-Activated Cell Sorting (FACS) Analysis can be used to detect specific combinations of cell surface proteins that may characterize the original blast population. For example, the lymphoid marker CD7 can be coexpressed along with CD33 or CD13 on blasts in a proportion of patients with AML, and this aberrant combination can be used as an immunologic "footprint" in follow-up studies.[5] Using FACS analysis, abnormal populations can be detected in as few as 0.1% (10-3) of cells.[1]

Technique

Marker

Detection Limits

FACS analysis

Antigen profile

10-3

Polymerase Chain Reaction (PCR) has emerged as the most promising approach to date for the detection of MRD. PCR offers the advantage of very high sensitivity with a detection limit of approximately 10-5.[1,6] This technique involves logarithmic amplification of specific deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences. For example, the PML-RAR-alpha protein can be amplified by PCR and can detect patients who, after therapy, are at risk for early relapse.[7] Alternately, in patients with t(8;21), the significance of a positive PCR result may be more complex.

The AML1-ETO rearrangement has been identified in patients even after long (less than 5 years) morphologic remissions.[8] In this unusual instance, the detection of MRD does not appear to be a prognostic factor.[9]

Technique

Marker

Detection Limits

PCR

DNA/RNA structure

10-5

Glossary of Genes

AML1-ETO--Acute myelogenous leukemia M1-Eight twenty-one[8]

EAP--Epstein-associated protein gene[13]

BCR-ABL--Breakpoint cluster regionóAbelson oncogene[10]

EVI-1--Ectopic viral integration site 1 gene[14]

CBFß-MYH11--Core binding factor-ß (smooth muscle) myosin heavy chain gene[11]

MDS--Myelodysplastic syndrome[10]

DEK-CAN--von Lindern and colleagues used patient DK to clone the hybrid CA(I)N, brother of Ab(e)l, as both are located on 9q34[12]

MLL--Myeloid-lymphoid leukemia or mixed-lineage leukemia1[15]

PML-RAR-alpha--Promyelocytic leukemia-retinoic acid receptor alpha[6]

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