1. CSI Case presentation by Khaled Alayed
2. The WHO Update Focused on Myeloid Neoplasms by Robert Paul HasserJian
3. The State of the Art of Acute leukemia of Ambiguous Lineage by Marie Christine Bene
4. Round Table: Approach to Acute Leukemia Testing by Brent Wood, Marie Bene and Sa Wang
The final plenary session at the ICCS meeting is named after the founders of the Society: Mariano LaVia and John Parker. This session included three presentations on the topic of acute leukemia and myeloid neoplasms, and one CSI case.
CSI Case 3
Presented by Khaled Alayed, with guidance from Howard Meyerson, Case Western University Hospitals, Cleveland.
Khaled described a 37 year old Hispanic male who presented with an acute febrile/viral syndrome and was found to have a hemophagocytic (HLH) syndrome secondary to Epstein-Barr virus (EBV) infection. He was treated with the HLH-94 protocol, but his fever persisted, blood counts dropped and he had atypical lymphocytic cells appeared in the peripheral blood. A bone marrow was performed which demonstrated an abnormal EBV positive lymphoid infiltrate. Flow cytometric immunophenotyping studies performed on the peripheral blood and bone marrow identified a population of cells with the following combined phenotype: CD2 positive, surface CD3 negative, CD5 negative, CD7 positive, CD8 negative, CD16 negative, CD56 positive, CD57 negative, CD25 negative, HLA-DR positive, CD71 positive, CD94 positive, CD161 negative and KIR antigen negative (CD158a1/a2 negative, CD158b1/b2 negative CD158e1/e2 negative). In addition, conventional cytogenetic studies demonstrated a complex abnormal karyotype. A diagnosis of aggressive NK-cell leukemia with hemophagocytosis was established, and the patient experienced the characteristic fulminant clinical course.
Khaled went on to summarize lymphoproliferative disorders of natural killer cells, as listed in the 2008 WHO classification. He then focused on immunophenotypic differences between activated NK cells, naïve NK cells, aggressive NK cell leukemia (ANKCL) and chronic lymphoproliferative disorder of NK cells (CLPD-NK) (Table 1), with reference to the following article: Lima M, et al. Leuk Lymphoma. 2015 Jan;56(1):103-12.
In summary, the main immunophenotypic characteristics of ANKCL cells (CD56 + bright, CD94 + bright, CD26+, CD16-/+ dim, CD57-, KIR-) appear to be similar to those of the CD56 + bright naïve NK cells present in normal PB. This immunophenotype is different from that found in chronic lymphoproliferative disorder of NK cells (CD56-/+ dim, CD94 + bright, CD16+, CD57-/+ dim, CD11c+ bright, KIR+). However, both of ANKCL and CLPD-NK can show an activation related phenotype (CD7-/+ dim,het, CD11b-/+ dim,het, CD45RO-/+ dim,het, HLA-DR-/+ dim,het)
WHO Classification Update Focused on Myeloid Neoplasms.
Robert P Hasserjian, MD, Massachusetts General Hospital and Harvard Medical School.
Rob Hasserjian gave an overview of the organization and rationale of the WHO Classification of Myeloid Neoplasms, reviewed current problems in the diagnosis and classification of myeloid neoplasms, and presented the 2016 revisions to the 2008 WHO classification (as outlined in the recent article from Arber DA et al., Blood 2016;127:2391). Rob gave a comprehensive summary of the categories of myeloid neoplasms, highlighted some of the newer molecular data, and mentioned the following areas pertinent to flow cytometric immunophenotyping:
1. Recognition of lymphocyte variant- hypereosinophilic syndrome associated with small phenotypically abnormal clonal T-cell populations detected by flow cytometry (CD3-CD4+ Th2 type cells). Therefore, flow cytometric evaluation of T-cells is recommended for patient with unexplained eosinophilia. (See the following article for a summary of the lymphocyte-variant hypereosinophilic syndrome: Boyer D. Arch Pathol Lab Med. 2016;140:1060–1067).
2. Identification of small lymphoblast populations in patients with at diagnosis of chronic myeloid leukemia. “However, because the onset of lymphoid BP may be quite sudden, the detection of any bona fide lymphoblasts in the blood or marrow should raise concern for a possible impending lymphoid BP, and prompt additional laboratory and genetic studies to exclude this possibility.”
3. Continuation of the recommendation that flow cytometric information alone is considered insufficient to establish a diagnosis of MDS, but can support a diagnosis suspected by morphology In the absence of morphological support, repeating bone marrow examination in 3-4 months is recommended.
4. Introduction of the new category of CMML-0 with < 5% BM blasts or < 2% PB blasts, with recognition of the difficulty often encountered in distinguishing promonocytes (blast equivalents) from mature monocytes by flow cytometry and morphology.
Rob concluded that the updated WHO Classification of myeloid neoplasms refines the definitions of many existing entities and introduces several new entities, but diagnosis and classification largely still rely on morphology. Some newer molecular data has been incorporated, but Rob emphasized that identification of a somatic mutation indicating clonal hematopoiesis does not necessarily imply the presence of a myeloid neoplasm, and a lot more work is needed to fully leverage this data in disease classification and treatment decisions. Let’s hope that by the time of the next revision of the WHO classification some of the challenges currently encountered with incorporating flow cytometric data in the diagnosis, classification and treatment decisions will be addressed.
The State of the Art of Acute Leukaemias of Ambiguous Lineage.
Marie Christine Bene, Nantes, France.
Marie Christine Bene started her presentation with an overview of acute leukemia and the approach to immunophenotyping proposed by European LeukemiaNet, including the proposed screening panel to identify lineage (see roundtable discussion below for further information). She them mentioned some of the difficulties encountered with lineage determination. The 2008 WHO classification introduced the term acute undifferentiated leukemia (AUL), when leukemia blasts lack lineage defining markers, and might in theory only express CD34 and HLA-DR. Marie Christine mentioned that in her experience AUL is rare, and perhaps as a consequence there is no recent literature. The second WHO category of acute leukemia of ambiguous lineage are those that demonstrate a mixed phenotype (MPAL), including cases with t(9;22) or t(n;11q23) and those not otherwise specified. MPAL encompasses the cases previously categorized as mixed phenotypic or mixed lineage acute leukemia. MPAL is also relatively rare, but might be under recognized when using limited immmunophenotyping panels. The 2008 WHO classification listed features of myeloid, B, and T lineage which have been modified slightly in the 2016 revision:
• Myeloid lineage: MPO, or bright myeloid markers CD117 CD13 CD33, or monoblastic/monocytic differentiation with NSE or >1 monocytic markers including CD11c, CD14, CD36, CD64, lysozyme
• B-lineage: CD19 and CD10 or CD19 and cCD79a, CD22, Pax5 (IHC)
• T-lineage: bright cCD3+/sCD3-
The 2016 revision also mentions the need to exclude CBF acute leukemia, acute promyelocytic leukemia and FGFR1 rearrangement, before making a diagnosis of MPAL. Marie Christine then reviewed some example cases and discussed the literature. Recent advances include identification of ADAMTS2 dysregulation (Tota et al, BMC Cancer 2014;14:963-8), the reported benefit of imatinib in Philadelphia chromosome positive MPAL (Shimizu et al, Eur J Haematol 2014;93:297-301) and identification of an association with DNMT3A mutations (Eckstein et al., Exp Hematol 2016 ;44:740-744). There is currently no consensus about therapy for MPAL, but use of acute lymphoblastic leukemia-like chemotherapy, plus or minus tyrosine kinase inhibitor therapy, possibly with AML-like salvage therapy and allogeneic stem cell transplant has been suggested (Wolach and Stone, Blood 2015;125:2477-2485).
Acute myeloid leukemia immunophenotyping roundtable discussion.
Marie Christine Bene, Nantes, France,
Brent Wood, University of Washington Seattle, USA,
Sa Wang MD Anderson, Houston, Texas, USA.
Fiona Craig from Mayo Clinic Arizona chaired the round table discussion, and started by asking each of the other participants to give a 5 minute presentation summarizing their approach to the flow cytometric evaluation of acute myeloid leukemia. Each included a summary of the panels used (Tables 2a, b and c). Fiona then summarized the key features of the different approaches (Table 3), and highlighted some of the differences with questions to the panel.
Tabe 2a: Sa Wang MD Anderson acute leukemia work up
Tabe 2b: Brent Wood, University of Washington Seattle acute leukemia workup
Tabe 2c: Marie Christine Bene, Groupe d’Etude Immunologique des Leucémies (GEIL) acute leukemia workup (10-color and 8-color).
Table 3: Comparison of approaches to acute myeloid leukemia immunophenotyping.
The panel agreed with Marie Christine that acute undifferentiated leukemia is uncommon. There was discussion about some confusion with interpretation of the 2008 WHO classification. In particular, a diagnosis of AML does not require myeloperoxidase expression, and can be established with expression of very few markers, such as CD13 and HLA-DR, usually with CD117 i.e. the list of features necessary to establish a myeloid phenotype in MPAL is not necessary to establish a diagnosis of AML.
There was a question from the audience about whether the term MPAL should be used for a therapy-related myeloid neoplasm or acute leukemia with MDS-related cytogenetic abnormalities. It was acknowledged that this is an area of confusion, and is somewhat arbitrary.
Fiona then questioned Brent on lack of upfront cytoplasmic evaluation. Brent mentioned that this strategy was currently under re-evaluation in the lab., and might change in the future.
The next question related to the different number of acquired events. Marie Christine emphasised that 10,000 – 30,000 events are only used for diagnosis, not post-treatment follow-up. Fiona inquired whether with fewer events a small population of mixed lineage might be over-looked. Brent mentioned that they routinely acquired 150,000 events to define smaller subsets and compare with the low frequency normal maturing precursors.
The audience then raised discussion about the criteria for early precursor T lymphoblastic leukemia, and whether cytoplasmic CD3 staining was necessary. The panel thought that it should be required but that is not specified in the WHO classification. Rob Hasserjain is going to investigate further and if possible include in the revised classification.
In summary, the roundtable discussion highlighted that different labs use different approaches and tried to clarify areas of potential misinterpretation of the WHO classification.