1. Acute Promyelocytic Leukemia
MLSCI 430
RabiaYousofi
Tino Villatoro

2. Our Case Study
A 29-year-old male was admitted to ER looking pale with a rash on his extremities.
He has had a persistent nose bleed for the last 2 hours.
Lab Values:
Hemoglobin87 g/L
Platelets15 x 109/L
WBC38 x 109/L
PT (INR)5.0
PTT62 seconds
Fibrinogen0.3 g/L
3. Further Lab Testing
A review of the peripheral blood smear revealed many abnormal cells.
A bone marrow aspiration and biopsy was later performed.
Samples were sent for flow cytometry, cytogenetics, and the molecular oncology lab.
A diagnosis of Acute Promyelocytic Leukemia (APL) was made on the basis of the tests performed.
4. What is Acute Promyelocytic Leukemia?
APL is characterized by the accumulation of blasts that are blocked at the promyelocytic stage of differentiation.
http://www.pnas.org/content/102/20/7174/F6.large.jpg
5. WHO vs. FAB
Acute promyelocytic leukemia falls under the old FAB classification as AML FAB M3 and M3v (for the microgranular variant).
Under the new WHO classification, APL falls under the category AML with recurrent genetic abnormalities.
This new classification recognizes the molecular/genetic feature of APL, namely the balanced translocation of chromosome 15 and 17.
6. PML-RARA: t(15;17)
The t(15;17) is characteristic and virtually diagnostic of APL.
This translocation results in the fusion of the PML gene on chromosome 15q22 and the RARA (Retinoic Acid Receptor A) on chromosome 17q21.
Expression of the PML-RARA protein results in a block in differentiation at the promyelocyte stage by suppressing RARA target genes.
7. Variant t(15;17)
There is a common breakpoint within intron 2 of the RARA gene and three breakpoints within the PML gene which results in the formation of three variants.
These breakpoints are:
Intron 6 (bcr1; 55% of cases)
Exon 6 (bcr2; 5% of cases)
Intron 3 (bcr3; 40% of cases)
Bcr3 is associated with M3v microgranular form. It has a higher incidence of DIC and a higher leukocyte count.
8. Variant APL Translocations
Other variant translocations may occur involving chromosome 17 that lead to APL:
t(11;17)(q23;q21)
t(5;17)(q35;q21)
t(11;17)(q13;q21)
der(17) (17q21.3-q23)
http://www.pathguy.com/lectures/m3.jpg
9. Variant Translocations
t(11;17) (q23;q21)
Most common and intensively studied variant
Fuses the PLZF gene (promyelocytic leukemia zinc finger) with RARA resulting in the expression of a PLZF-RARA protein.
Falls into an unusual morphologic spectrum of APL,with features intermediate between M2 (AML with some maturation) and M3 (APL).
Important to recognize, as this translocation is insensitive to ATRA
10. Variant Translocations
t(5;17) (q35;q21)
Second-most common variant
This variant translocates the nucleophosmin gene on 5q35 into the RARa locus on 17q21
Nucleophosmin is a nucleolarphosphoprotein that plays a role in ribosomal RNA assembly; it also has chaperoning activities, as well as nuclease activity.
The phenotype is identical to APL M3
In-vitro studies have shown that promyelocytes of t(5;17) are still sensitive to ATRA, and this has been shown in one case study as well.
11. Variant Translocations
t(11;17)(q13;q21)
This is a rare APL variant
Blood smear and bone marrow specimens show a predominance of promyelocytes and dysplastic maturing neutrophils.
This variant is still sensitive to ATRA
der(17) (17q21.3-q23)
Morphologically similar to AML M1 (AML with minimal differentiation) with aminority of marrow blasts showing morphologic evidence for the M3v microgranular variant of APL.
Shows no response to ATRA
12. Cytogenetic Diagnosis
Cytogenetic studies can reveal the abnormal karyotype in APL.
Several banding techniques are available, including giemsa-trypsin banding, r-banding, and c-banding.
Metaphase chromosomes are treated with trypsinand stained with Giemsa. This creates a unique banding pattern for each chromosome.
13. http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cmed&part=A1548&rendertype=figure&id=A1551
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cmed&part=A1548&rendertype=figure&id=A1557
14. Fluorescent In-Situ Hybridization
http://en.wikipedia.org/wiki/File:FISH_%28Fluorescent_In_Situ_Hybridization%29.jpg
15. Minimal Residual Disease
Quantitative reverse transcriptase PCR can be used to detect the PML-RARA transcript.
Patients with a higher transcript level tend to have a worse prognosis.
Treatment and remission can also be monitored using real-time quantitative RT-PCR.
16. 17. Typical Features of Blast Cells
Myeloblast
Monoblast
Proerythroblast
Megakaryoblast
Lymphoblast
18. Myeloblast
< 1% of the normal bone marrow, not observed in normal blood
Vary in size, but are usually large
Nucleus is delicate, large, round or Sl oval, with prominent nucleoli. Stain purplish red with Wright stain. Chromatin stains evenly
Small to moderate amount of bluish nongranular cytoplasm
Three major types: Type I, II, and III
19. Type I
Fine nuclear chromatin
2 to 4 distinct nucleoli
Moderate rim of pale to basophilic cytoplasm
Without azurophilic granules
Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 8).
20. Type II and III
Type II
Nuclear and cytoplasmic features are similar to type I
Delicate azurophilic granules in the cytoplasm (up to 20)
Type III
Numerous azurophilic granules in the cytoplasm
Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 9).
http://www.pathologyoutlines.com/leukemia.html
21. Auer Rods
One characteristic feature of myeloblasts in AML
Presence of Auer rods with abnormal azurophilic granules
(60% - 70% of all cases)
(faggot cells)
Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 10).
22. Monoblast
Derived from myelocytic-monocytic progenitor cells in BM
Round, sometimes folded, large early nucleus with 1 or 2 nucleoli
Finely dispersedlinear chromatin
Small indentation (nuclear creases)
Basophilic cytoplasm with no granules (sometimes fine granules & occasional vacuoles)
23. Erythroblast
0% - 1% in normal bone marrow of adults
Round nucleus, Sl condensed nuclear chromatin
Variable prominent nucleoli
Moderate amount of deeply basophilic cytoplasm.
24. Megakaryoblast
Moderately large cell with 1 or 2 nuclei (round nucleus)
Nucleus begins to indent with lobes start to form and nucleus increases in size
Nucleoli often demonstrable
Non-granular cytoplasm, may have blunt pseudopods or blebs, stain bluish
In AML, very significantly in appearance from one case to another
25. Promyelocyte
http://www.healthsystem.virginia.edu/internet/hematology/hessidb/normal-hematopoietic-cells.cfm
Primary granules (azurophilic or dark blue), no secondary granules
1% - 5% in normal bone marrow, not seen in normal blood
Its size varies (may exceed 20m)
Nucleus is round and large in relation to the cytoplasm
Chromatin is sl courser than myeloblast
Nucleoli may be visible (often indistinct)
Cytoplasm is dark blue with a relatively light area adjacent to the nucleus
26. NeoplasticPromyelocyte (Blast Equivalent)
Eccentric, often folded and lobulated nucleus
Sl condensed nuclear chromatin
Intense cytoplasmicgranulaity
An apparent Golgi zone
Konoplev S et al. Advances in the pathologic diagnosis and biology of acute myeloid leukemia (figure 10).
27. Cytochemical Stains
Since the early 20th century, cytochemical staining of cells has been a useful tool in differentiating hematopoietic diseases.
Smears and imprints made from bone marrow, lymph nodes, spleen, or peripheral blood are preferred.
In enzymatic techniques, fresh smears are used to ensure optimal enzyme activity
Certain elements may be inhibited during the fixation of smears and imprints
28. Myeloperoxidase (MPX/MPO)
The proxidase enzyme reacts with H2O2 & release O2, which oxidizes the indicator dye and produce orange-brown granules in the cells (3-amino-9-erythrocarbazol)
Enzyme MPX is found in the 1o granules of granulocytes, neutrophils and precursors (from the promyelocyte stage on) & eosinophils
Monocytes may be weakly pos
Leukemic myeloblasts are usually pos and Auer rods stain very strongly
Used for differentiating AML (+) from ALL (-)
Normal bone marrow smear