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

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

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.

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)

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.

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.

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.

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]

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.

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

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

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) http://ghr.nlm.nih.gov/gene/TP53

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): 1539-1541 Blood 2012 (119): 2114-2121 Leukemia & Lymphoma 2012 52(2): 336-337

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 http://p53.iarc.fr/

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): 656-663 Blood 2012 (119): 2114-2121

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): 642-647

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