Myeloid sarcoma or mixed phenotype acute leukemia? Multiparametric flow cytometry to the rescue in an unusual diagnostic dilemma

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Myeloid sarcoma (MS) is an uncommon presentation of myeloid neoplasm, resulting from the infiltration and effacement of tissue architecture by leukemic blasts at an anatomical site other than bone marrow. The most common sites of involvement are the skin, lymph nodes, soft tissue, bones, gastrointestinal tract, and so on. These neoplasms express a wide array of myeloid lineage markers. Mixed-phenotype acute leukemia (MPAL) is an uncommon hematolymphoid neoplasm showing proliferations of blasts with immunophenotypic features of two (or more) lineages, usually myeloid lineage along with either T or B lineages. MPAL presenting as MS (extramedullary mass, without leukemic presentation) is extremely rare; less than 20 cases have been reported in the literature so far (Means et al., 2022). We report one such case, where multiparametric flow cytometry (MFC) evaluation for lineage assignment was especially useful due to its ability to detect a large array of antigens on particular cell populations, and helped clinch the diagnosis. Differentiating MS (having only myeloid lineage blasts) from MPAL is extremely important since treatment strategy and prognosis varies (Figure 1). A 37-year-old gentleman presented with multiple neck swellings for the past 8 months. The complete blood count showed a hemoglobin of 158 g/L, total leukocyte count of 10.14 × 109/L, and platelet count of 327 × 109/L. A peripheral smear examination did not reveal any atypical cells or blasts. Liver function tests and renal function tests were normal. Lactate dehydrogenase was 268 U/L, which was mildly elevated (the reference range was 0–248 U/L). By ultrasonography, liver and spleen appeared normal in size and echotexture. A biopsy of the supraclavicular lymph node revealed effacement of nodal architecture by atypical blastoid cells. The cells were found to be positive for myeloperoxidase on immunohistochemistry, while CD3 and CD20 were reported to be positive in the background T-cells and B-cells respectively. These findings led to the diagnosis of myeloid sarcoma. A bone marrow examination was performed to determine if there was infiltration. Bone marrow aspiration revealed 16% blasts on morphology. Flow cytometric immunophenotyping of the bone marrow aspirate showed that 16% of all viable events were in the blast region (CD45 dim, low-side scatter). Approximately 20% of these (amounting to 3.2% of all viable events) were abnormal blasts expressing CD34, HLA-DR, CD13 (dim), CD33, CD117, and MPO. In addition, these blasts also showed positivity for T-lineage markers CD5 (dim), CD7, CD56 (dim), and cytoplasmic CD3. The remaining events in the blast region (~12% of all viable events) were haematogones. Since the blasts were expressing both T- and myeloid lineage markers (including the lineage defining CD3 and MPO), we concluded that bone marrow was infiltrated by abnormal blasts showing a mixed phenotype (T/myeloid). In view of the flow cytometry findings, we also realized that the 16% blasts on morphology possibly included many hematogones, and the true extent of bone marrow involvement by the leukemic blasts was likely much lesser. The discrepancy between the findings from the lymph node biopsy and bone marrow aspiration (myeloid sarcoma vs. presence of mixed phenotype blasts) prompted the fine needle aspiration of the lymph nodal mass for flow cytometry, which revealed the presence of 57% blasts (CD45 dim, low side scatter) which expressed CD34, HLA-DR, CD38, CD13, CD33 (bright), CD117 (dim), CD64, and MPO, along with surface CD3 (dim), CD5 (dim), CD7, and cytoplasmic CD3. This confirmed the presence of mixed lineage blasts (T/myeloid) in the myeloid sarcoma. Molecular studies for BCR::ABL1 rearrangement and MLL::AFF4 rearrangement were negative. The patient was started on a modified BFM (ALL-type) treatment protocol. At the end of induction, the patient was doing well and his neck swelling had disappeared. End-of-induction bone marrow examination revealed only 3% blasts by morphology, but 0.31% residual leukemic cells were noted on flow cytometry based measurable residual disease (MRD) assay. At the end of 8 weeks of therapy, bone marrow was in morphological remission, and flow cytometry based MRD was negative. Subsequently, an allogenic hematopoietic stem cell transplant has been planned for the patient, and he is being worked-up for the same. MPAL is a rare leukemic entity, accounting for less than 5% of all leukemias. It consists of either a single population of blasts expressing markers of two lineages (biphenotypic), or two abnormal populations of blasts of different lineages (bilineage). Immunophenotypic criteria for lineage assignment in cases of MPAL have been revised and updated in 5th edition of World Health Organization classification of haematolymphoid tumors (Khoury et al., 2022). Extramedullary presentation of MPALs without leukemic involvement is very unusual; B/myeloid MPAL is the most common type in these MS-like presentations, while T/myeloid and B/T MPAL are relatively rarer (Means et al., 2022). Our case has mixed phenotype (T/myeloid) blast proliferation in lymph nodes; these abnormal blasts were absent in peripheral blood, and constituted only 3.2% of all cells in the bone marrow aspirate (by flow cytometry). Such cases should not be labeled as MS, as MS shows proliferation of blasts which express only myeloid lineage markers, and cases which fulfill the criteria of MPAL are excluded from this category of tumors. These cases pose a significant diagnostic challenge, and can easily be misdiagnosed as small blue round cell tumors, lymphoblastic leukemias or AML, especially if the panel of markers used for immunohistochemistry (IHC) is limited. Even in the presence of adequate markers on IHC, the interpretation can be very challenging, given the rarity of these neoplasms. MFC-based analysis of fine needle aspirate from these tumors is an extremely valuable tool for diagnosing these rare neoplasms, as beautifully demonstrated in this particular case, because MFC has very high sensitivity, and a larger panel of markers can be simultaneously studied in a cell population. We stress the importance of making a correct diagnosis, since the prognosis of MPAL is considered poor compared to de novo AML or ALL (Khanna et al., 2017). In addition, there are reports that MPAL patients receiving an ALL-based chemotherapeutic regimen achieved higher remission rates than those receiving an AML-based regimen (Tian et al., 2016). However, it is important to note that optimal induction chemotherapy for MPAL has not yet been well-established, and larger studies of AML- and ALL-based regimen incorporating genetics and immunophenotype is warranted. To conclude, we present an unusual case showing mixed phenotype (T/myeloid) blast proliferation in lymph nodes without overt leukemic presentation, which was initially misdiagnosed as MS. This case highlights how flow cytometry-based evaluation can be crucial in reaching a correct diagnosis of this rare presentation of MPAL, thus helping in proper prognostication and treatment. The authors would like to thank Dr. Aravind Sekar for providing inputs on the lymph node biopsy findings. The authors declare no conflicts of interest. The patient data is available on request from the corresponding author.

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