Submitting Author: Al-Ibraheemi, Alyaa, MD
Institution: MD Anderson Cancer Center
Additional authors:Roberto N. Miranda, M.D., Sa Wang, MD, Carlos E Bueso-Ramos, MD, PhD, Jeffrey L. Jorgensen, MD, PhD
Session: AML with recurrent genetic abnormalities Part I

31-year-old male, presenting with peripheral blood leukocytosis including 67% monocytes and 28% blasts. A bone marrow biopsy and aspirate (Specimen 1) led to a diagnosis of acute monocytic leukemia with MLL gene rearrangement. Flow cytometric immunophenotyping at diagnosis showed a distinct blast population mostly in the monocyte region, notably positive for CD14, CD33 bright, CD34, CD64 bright, and CD117 (small subset only). Conventional cytogenetic studies at diagnosis showed a pseudodiploid clone: 46, XY, del (11)(q23). The patient received chemotherapy with clofarabine, idarubicin, and cytarabine. His post-induction course was notable for neutropenic fever, with pnemonia and Enterobacter bacteremia. He did not receive exogenous growth factors.

On day 20-post induction therapy, his bone marrow aspirate smear (Specimen 2) showed 21% blasts, 14% progranulocytes, 15% myelocytes, 18% metamyelocytes, 10% granulocytes, and 5% monocytes. Flow cytometric immunophenotyping showed markedly left-shifted myeloid maturation, with an unremarkable spectrum of maturation markers. Conventional karyotyping showed a diploid male karyotype, and FISH studies were negative for the presence of a clone with MLL gene rearrangements.

A subsequent bone marrow aspiration on day 29 (Specimen 3), with no intervening chemotherapy, showed a cellular marrow with 5% blasts and unremarkable complete myeloid maturation. He received a second course of therapy using the same chemotherapeutic agents. A bone marrow aspiration on day 90 showed orderly trilineage maturation. Both subsequent specimens showed a diploid male karyotype, negative by FISH for the presence of a clone with MLL gene rearrangements.

On 11/20/2012 he underwent an allogeneic stem cell transplant from a matched unrelated donor on protocol 2011-0628 with fludarabine and IV busulfan conditioning. The post transplant course has been uncomplicated and the patient is doing well at last follow up, with no evidence of residual AML.

Specimen 1

Bone marrow biopsy site: Right posterior iliac crest

Method of fixation: 10% buffered-formalin

Gross measurement: 1.5, 0.3 cm

Specimen 2

Bone marrow aspiration site: Right posterior iliac crest

Method of fixation: 10% buffered-formalin (clot preparation only)

Specimen 3

Bone marrow aspiration site: Left posterior iliac crest

Method of fixation: 10% buffered-formalin (clot preparation only)

Microscopic description

Specimen 1:

Hypercellular marrow (95-100%) with decreased trilineage hematopoietic cells; infiltrated by sheets of blasts that were intermediate to large in size with moderately abundant cytoplasm.

BM aspirate smear revealed 55% blasts, and 35% monocytes.

The blasts were negative for myeloperoxidase (cytochemical stain). 80 % of the blasts were positive for butyrate esterase (cytochemical stain).

CBC revealed Hgb 8.7 g/dL, plt 143 x 109/L and WBC 94.8 x 109/L with 2% neutrophils, 3% lymphocytes, 67% monocytes and 28% blasts

Specimen 2:

Cellular marrow (60-70%) with decreased trilineage hematopoietic cells; infiltrated by immature cells that are intermediate to large in size with moderately abundant cytoplasm.

BM aspirate smear revealed 21% myeloid precursors, 14% progranulocytes, 15% myelocytes, 18% metamyelocytes, 10% granulocytes, and 5% monocytes.

CBC revealed Hgb 9.1 g/dL, plt 36 x 109/L and WBC 0.6 x 109/L with 20% neutrophils, 47.5% lymphocytes, 27.5% monocytes and 5% blasts

Specimen 3

Cellular marrow (60-70%) with trilineage hematopoietic cells; granulocytes showed progressive maturation. Scattered immature cells were noted.

BM aspirate smear revealed 5% myeloid precursors, 1% progranulocytes, 12% myelocytes, 5% metamyelocytes, 24% granulocytes, and 7% monocytes.

CBC revealed Hgb 10.4 g/dl, plt 604 x 109/L and WBC 4.1 x 109/L with 69.0% neutrophils, 9% lymphocytes, 17% monocytes and 2% blasts

Specimen 1

By flow cytometry the blasts could be gated in a combined CD45 dim/CD45 bright mononuclear gate, comprising the blast and monocyte regions on a CD45/SSC plot. They were positive for: CD13 partial, CD14 variable, CD33 bright, CD34 variable, CD38, CD56 (subset), CD64 bright, CD117 (small subset), CD123, and HLA-DR. The blasts were negative for: CD2, cytoplasmic CD3, CD5, CD7, CD10, CD19, myeloperoxidase, and TdT.

Specimen 2

By flow cytometry there was a distinct population of CD34+ myeloid precursors, which in comparison with the original blasts showed significantly greater expression of CD117, and were negative for CD14 and CD64. They showed a spectrum of maturation markers which matched those in normal bone marrow aspirates.

Specimen 1: Pseudodiploid clone 46, XY, del (11)(q23) [13], diploid male karyotype (46,XY[7]) ; negative for BCR/ABL1 and MYC gene arrangements by FISH.

Specimen 2: diploid male karyotype (46, XY [20]); negative for the presence of a clone with MLL gene by FISH

Specimen 3: diploid male karyotype (46, XY [19]), 46,XY,t(6;17)(q21;q25)[1] ; negative for the presence of a clone with MLL gene by FISH. The single abnormal metaphase was regarded as not representing clonality as far as official cytogenetic nomenclature. It did not persist in a subsequent bone marrow aspirate on day 90.

Specimen 1 : Negative BCR-ABL fusion transcript by real-time PCR; negative for FLT3 (ITD and codon 835/836 mutation), and negative by PCR for RUNX1-RUNX1T1, inv(16)(p13.1q22) or t(16;16)(p13.1q22); CBFB-MYH11 variant E, t(15;17)(q22;q12); PML-RARA long form, t(15;17)(q22;q12); PML-RARA short form, t(9;22)(q34;q11.2); BCR-ABL1 b2a2, t(9;22)(q34;q11.2); ETV6-RUNX1, t(1;19)(q23;p13.3); and t(4;11)(q21;q23); MLL-AF4.

Specimen 2: not performed

Specimen 3: not performed

Traditional morphologic criteria for remission in acute myeloid leukemia rely on a threshold of 5% blasts. However, during early marrow recovery from ablative chemotherapy or pre-transplant conditioning, a markedly left-shifted spectrum of myeloid maturation can be seen. In occasional patients, this can include remarkably large numbers of morphologic blasts, here up to 21%. Under the influence of endogenous and/or exogenous growth factors (e.g., G-CSF therapy), the immature myeloid and monocytic cells can show atypical morphology, making them very difficult to distinguish from residual leukemic blasts.

Ancillary techniques to detect residual disease in AML primarily include flow cytometry, cytogenetic studies including FISH, and molecular assays for recurrent gene rearrangements and mutations. Much of the focus in the literature has been on sensitive detection of minimal residual disease (<5% of total cellularity). This case illustrates the additional capability of flow cytometry to rapidly rule out persistent AML in early marrow recovery, in cases with increased morphologic blasts. In this case, the flow cytometry diagnosis of normal marrow recovery was supported by subsequent karyotypic and FISH studies, as well as the clinicial course, with rapid improvement in peripheral blood counts and a marrow aspirate 9 days later showing normal trilineage maturation.

Normal surface antigen expression during differentiation is uniform and tightly regulated, and thus normal cells demonstrate characteristic antigen expression patterns that are reliably observed in healthy individuals. Leukemic blast cells display inappropriate cell surface antigen patterns, that may differ significantly from normal expression patterns. Therefore blasts can in most cases be distinguished by flow cytometry from normal hematopoietic elements. Familiarity with normal antigen expression patterns on maturing myeloid precursors, in both “resting” and recovering marrow, is key to interpreting flow cytometry data in AML residual disease assessment.

Specimen 1: Acute monocytic leukemia with MLL gene rearrangement

Specimen 2: Recovering marrow demonstrating marked left-shifted granulopoeisis, including 21% blasts

Specimen 1: Acute myeloid leukemia with MLL gene rearrangement

Specimen 2: Recovering marrow demonstrating marked left-shifted granulopoeisis

Case 417