1. AML with Myelodysplasia-Related Changes Case 374
Beth Chastain, MD
University of New Mexico
No financial disclosures

2. Clinical and initial laboratory data
65-year-old male presenting with weakness, dizziness, and epistaxis intermittently for one month
CBC
WBC 35.6 x 103/mm3
Hgb 11.2 gm/dL
Plt 38 x 103/mm3
Differential shows 24% blasts

3. Peripheral blood smear
Circulating blasts (black arrow at top) are intermediate to large in size with scant to moderate cytoplasm, high N:C ratio, fine chromatin and prominent nucleoli. A subset of blasts have fine cytoplasmic granules. Maturing granulocytes show significant dysplastic features including hypogranularity and hypolobation (red arrow heads), including pseudo Pelger Huet cells, some of which contain cytoplasmic vacuoles. This morphologic feature has been associated with TP53 deletions.

4. Peripheral blood smear
Another PB field again demonstrating the presence of a circulating blast and hypogranular and hypolobated maturing myeloid cells, including pseudo-Pelger-Huet forms (black arrow)

5. Bone marrow aspirate
BMA demonstrates an increase M:E ratio, ~22% blasts (black arrows), maturing granulocytic precursors with hypolobated nuclei including pseudo-Pelger-Huet forms (red arrow heads). Erythroid precursors exhibit dysplastic changes including binucleation, abnormal hemoglobinization, and basophilic stippling (double green arrows). Megakaryocytes are exceedingly rare.

6. Bone marrow biopsy
Bone marrow core biopsy and clot section show essentially 100% cellularity composed predominately of left-shifted myeloid precursors. Hypolobation is the dominant feature within the maturing cells. Scattered erythroid precursors are seen and rare megakaryocytes demonstrate hypolobation.

7. Flow cytometric analysis
Blast population (red):
Positive for:
CD34
CD117
CD13
HLA-DR
Subset CD33 (not shown)
Subset CD4 (not shown)
Negative for:
CD45
Monocytic markers (CD14, CD64, CD36, CD15)
Additionally, granulocytes are shifted down on side scatter secondary to the hypogranularity.

8. Karyotype Complex karyotype with multiple sidelines:
45, XY, der(16)t(16;17)(q12~13;q11.2), -17 [3]
46, sl, +11 [10]
47, sdl1, +9, +11 [2]
47, sdl2, +11, +13 [2]
46, XY [3]

9. FISH
FISH using a break apart probe for core binding factor beta gene demonstrates no rearrangement; however, there is deletion of one copy of CBFB gene in 86% of cells.

10. FISH
FISH for TP53 gene confirms deletion of one copy within the majority of cells.
FISH: TP53 gene

11. Diagnosis AML with myelodysplasia-related changes
Multilineage dysplasia
Complex karyotype with del TP53

12. TP53 Tumor protein p53 Chromosome 17p13.1
Protein acts as a tumor suppressor, regulating cell division and apoptosis
Somatic mutations are among the most common genetic changes in cancer
Majority occur within the sequence-specific DNA-binding domain (exons 4-8)

13. Alterations of TP53 in AML
Rare, 5-10% overall, as sole abnormality <1%
Associated with complex/monosomal karyotypes
10-15% of adult AML have CK
~70% of CK will have TP53 alteration
Patients tend to be older on average and have lower BM blast counts
Definition of CK not entirely standard: most consider >3 abnormalities and a few consider >5 abnormalities. Monosomal karyotype = two autosomal monosomies, or one autosomal monosomy associated with at least one structural abnormality. Most AMLs that fulfill criteria for CK also fulfill criteria for MK (~75-80%). Recent studies show that monosomal karyotypes confer a worse prognosis than complex karyotypes.
Age: median 61 vs. 54 (those with CK and TP53 mutations compared to those with CK/-TP53)
Blast count: median 65% vs. 78%
Leukemia 2008 (22):
Blood 2012 (119):
Leukemia & Lymphoma (2):

14. Types of abnormalities
TP53 Loss
del 17p
-17
No TP53 Loss
Compound heterozygote
Homozygous
Easily identified by karyotype analysis and FISH studies
TP53 loss more common than no loss (~60% vs. ~40%; with high mutation rate within the remaining allele). Of those without loss, the cmpnd hets are more common that the homozygous. Effect of abnormalities without complete loss of an allele depends on the type of mutation and the resulting protein, if any, formed. The International Agency on Research on Cancer (IARC) has a great website to search TP53 alterations and predicts protein function. No difference on outcomes between the different abnormalities.
-Missense
-Indels
-Nonsense
-Splice Site
Requires array-based or sequencing methods

15. Predictive and prognostic implications
Resistance to chemotherapy
Decreased response to induction
Fewer with complete remission
Inferior survival/dismal outcome (compared to CK+/TP53-)
Event-free: 1% vs 13%
Relapse-free: 7% vs 30%
Overall: 3% vs 28%
CR: ~30-35% with complex
Leukemia 2009 (23):
Blood 2012 (119):

16. Testing for TP53 alterations
Deletion:
Typically initially identified on karyotype (del 17p and -17)
Confirm with FISH
Perform FISH on CK/MK even if no initial TP53 mutation identified by karyotype
Without deletion:
Array based or sequencing methods not initially performed in clinical cases
Can be pursued in CK; however CK alone is poor prognosis
*some losses of TP53 can be identified with FISH that were not present on conventional karyotype analysis
ClinicalTrials.gov: numerous clinical trials are underway in many cancer types to determine effects of TP53 therapies but currently none are looking at AML
Leukemia & Lymphoma, April 2001;52(4):

17. Thank you UNM HSC and Presbyterian Hospital Qian Zhang, MD, PhD
Alexei Bakhirev, MD
Fred Elder, PhD
Cytogenetics lab