Plasmacytoid Dendritic Cell Proliferation Associated with Acute Myeloid Leukemia, Case Report and Review of Literature

    

    

    Figure 3: AML MRD study on a one-month post-chemotherapy bone marrow sample. A population of pDCs was measured at approximately 0.2% of total cells, based upon the positive CD123 and positive HLADR percentage. A subset of pDCs expresses dim CD34. Myeloblasts were present at approximately 0.4% of total cells, based upon the CD34 positive percentage. Subsets of blasts express dim CD11b, dim subset CD7, and dim CD3d (histogram not shown). Lower limit of detection: 0.01%.
    

Discussion and Review of Literature

Plasmacytoid Dendritic Cell Development and Summary of Reported Pdc-AML Cases

DCs are innate immune cells that initiate and control the response of the human immune system [5]. The classification of DCs has evolved from an anatomic classification based on where they reside to a lineage differentiation classification correlating with transcription factor expression. Currently, pDCs are defined primarily by expression of interferon regulator factors 8 and 4 (IRF 8 and 4), whilst classic or conventional DCs (cDC) lack either IRF4 or IRF8 including myeloid DCs and monocyte- derived DC (mo-DC) [5]. Historically, pDCs are thought to have a myeloid origin, but recent research argues the lymphoid origin of the pDC, based on several single-cell sequencing study results [6]. The origin of pDC and its development is still debated.

As early as 2009, Martin-Martin et al. [7] described three different maturation stages of pDCs found in normal bone marrow using markers including CD34, CD123, CD45 and HLADR. The pDC maturation stages were primarily based on the presence and absence and the expression intensities of these markers. The different pDC maturation stages were also studied by Huang et al. in 2020 on bone marrow samples from 14 patients diagnosed with pDC proliferation in myeloid neoplasm [8]. pDC proliferation in tissue in AML patients has been sporadically reported. In 2010, Dargen et al. reported pDC proliferation in the skin of a patient with AML [9]. Those pDCs did not express immature markers such as CD34. In 2012, Song et al. also reported a case of tumorous proliferation of pDC and Langerhan cells in a lymph node in a 55-year man with AML. Again, pDCs in that patient had a mature phenotype, corresponding to stage III pDCs. pDC proliferation in bone marrow was first reported by Wang et al. in 2017 [10]. It is unclear whether mature pDC proliferation in tissue and bone marrow in the context of AML are the same neoplastic process as those in pDC-AML. Reports have shown that pDCs in MPDCP associated with AML had genetic mutations and expression profiles similar to those in the associated AML blasts, including pathogenic RUNX1 and FLT3-ITD mutations [11]. The following table summarizes reported cases where pDCs were increased in bone marrow with a concurrent diagnosis of AML. Cases of bone marrow mature pDC proliferation associated with AML are also included in this table.

Differential Diagnosis with BPDCN and MPDCP Associated with other Myeloid Neoplasms

Our case is typical of pDC-AML with the characteristic flow cytometry immunohistochemistry phenotypic features. pDCs were readily identified by high expressions of CD123 and HLADR, and the immature nature of the pDCs was demonstrated by dim CD45 and dim CD34 expressions. CD303 expression, a specific marker for pDCs, performed by immunohistochemistry, confirmed the pDC proliferation. It is important to differentiate pDC-AML from BPDCN and Mature Plasmacytoid Dendritic Cell Proliferation (MPDCP) associated with other myeloid neoplasms since treatment for these disorders is quite distinct.

BPDCN is a distinctive entity recognized by the WHO since 2008 under plasmacytoid dendritic neoplasms [4]. The International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias on BPDCN remains unchanged, while pDC-AML is not listed by the ICC. BPDCN arises from pDC precursors with a clinical presentation different from that in pDC-AML. Virtually all patients with BPDCN present with skin lesions, as well as lesions commonly involving the lymph nodes and bone marrow. Morphologically, the neoplastic cells are small or intermediate sized, often with a moderate amount of cytoplasm and an eccentric nucleus, and prominent nucleoli. Neoplastic cells in BPDCN express pDC-associated makers CD123, CD303 and TCF4. CD123 is usually expressed at high level, compared with low-level expression in other hematologic diseases, including AML. BBDCN cells also express CD4, CD56 and TCL1 and sometimes aberrantly express CD2, CD5, CD7, CD33, CD38, CD68, CD117, HLADR and TdT [12]. Immature markers such as CD34 have not been reported in BPDCN but frequently seen in pDC-AML. pDCs in pDC-AML have maturation continuation from the myeloblasts. Gene mutations in BPDCN have been reported involving TET2, ASXL1, NRAS and ATM, while mutations in pDC-AML predominantly involve RUX1. Patients with BPDCN have historically been treated with systemic chemotherapy regimens like those used for AML, but more recently the approval of tagraxofusp [13] (a CD123-directed cytotoxin) for this population has resulted in significantly improved rates of remission success and allows many patients to transition to allogeneic transplant with curative intent. CAR-T therapy has also been used [14] to treat BPDCN, but data on pDC-AML treatment are still limited.

MPDCP associated with other myeloid neoplasms are clonal proliferations of pDCs, often seen with a nodular growth pattern in bone marrow, with low grade morphology. These mature pDCs express the normal phenotype with normal levels of CD123, CD303, CD304 expression, but without CD56 or CD34 and negative for TdT or CD2 or CD7 [15]. The associated myeloid neoplasm may be myeloproliferative CMML, MDS, MDS/MPN or AML, harboring activating RAS pathway mutations [4].

Implications for Flow Cytometry AML MRD

Flow cytometry AML MRD evaluation is an important part of diagnostic and prognostic evaluation. Many studies have shown that a positive flow cytometric MRD after induction and/or consolidation is correlated with an increased risk of relapse and worse survival [16]. The presence of MRD prior to allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is an important predictor of relapse and survival [17]. MRD status can be used for risk stratification and clinical decision making for patients with AML. Multi-color flow cytometry, in conjunction with molecular and cytogenetic studies, are probably the most used approach to detect MRD in AML.

MRD detection by flow cytometry has a sensitivity of approximately 10^-3 to 10^-4 (0.1 to 0.01%). The interpretation of flow cytometry AML MRD, however, is still expert-based and the interpretation of AML MRD for pDC-AML is especially challenging for several reasons. Due to the rarity of pDC-AML and BPDCN, AML MRD panels currently employed in many institutions do not include pDC-specific markers, such as CD303. In addition, there is limited literature on AML MRD evaluation on BPDCN, restricting the dissemination of knowledge to experts. Wang et al. has developed an one-tube assay to evaluate MRD for BPDCN [18]. The one tube assay includes 10 CD markers: CD2, CD7, CD38, CD303, CD123, HLADR, CD64, CD4, CD45 and CD56. It has been validated to a sensitivity of 0.01%. They also reported that the differentiation between reactive pDCs and neoplastic pDCs can be challenging and largely relies on CD56 expression. Since pDCs in pDC-AML carry the same mutation as the myeloblasts (or stage 1 pDCs), one could argue that the MRD evaluation in pDC-AML should include the evaluation of both myeloblasts and the immature pDCs, rather than myeloblasts alone. Moreover, the monocytes in pDC-AML also have the same mutation as the myeloblasts and immature pDCs and are phenotypically abnormal. We observed aberrant CD2 and CD5 expressions on the monocytes in the initial bone marrow in our case. Should the evaluation of mature monocytes also be included in the AML MRD evaluation?

Detection of MRD by flow cytometry is achieved by detection of immunophenotypic aberrancies on leukemic blasts or blast equivalents by using either one or both of two partially overlapping analysis strategies: focusing on “Leukemia-Associated Immunophenotypes” (LAIP) detected at the time of diagnosis, and identifying any immunophenotypes which are “different-from-normal” in specimens submitted for MRD analysis. Like many others, we employ a strategy using a “different-from-normal” approach, in combination with the knowledge of the diagnostic LAIPs when available. In approximately 90% of AML patients, leukemic blasts at diagnosis show aberrant expression patterns of one or more LAIP antigens which differentiate them from normal myeloid precursors and other marrow cells. In our case, myeloblasts had aberrant CD7 and CD56 expressions that can serve as MRD evaluation for the follow up samples. Detecting MRD based only on LAIPs identified at the time of diagnosis can lead to some problems due to “immunophenotypic shift” at relapse that can occur in around 90% of AML [19]. The frequent antigenic drift or immunophenotypic switch during AML treatment makes LAIPs a “moving target” for MRD detection. “Different from-normal” is used to distinguish abnormal leukemic blasts from normal myeloid precursors irrespective of diagnostic LAIPs. Normal hematopoietic precursors show a tightly sequenced and predictable maturation pattern which can be visualized by flow cytometry. The knowledge of “normal expression,” usually obtained with a comprehensive control group including both normal and regenerating bone marrows, run in the same laboratory with the same MRD panel. In our case, the AML MRD performed one month post chemotherapy detected a population of immature pDCs (0.2% of total cells), based upon the CD123 positive and HLADR positive percentage, and these pDC appears to be immature with a subset expression of dim CD34. Further evaluation on these immature pDCs was limited due to the panel of CD marker selection in our AML MRD panels. The myeloblasts (0.4% of total cells) did not other display phenotypic aberrancy except dim subset CD7 and dim CD36. The determination of residual pDC-AML is difficult in this case. The minor phenotypic changes in the myeloblasts are considered likely phenotype shift related to chemotherapy.

Traditional molecular methods, such as quantitative PCR (qPCR) for AML MRD, rely on the molecular targets that can be amplified; over 40% of patients do not have an identifiable PCR target. NGS has recently been used to detect AML MRD gene mutations, and more than 90% of AML patients carry molecular alterations that serve well for NGS detection [20]. Using error corrected NGS, Thol et al. [20] and Patkar et al. [21] significantly increased the sensitivity of AML MRD detection. Press et al. [22] studied 42 AML patients using NGS method with a LLOD 0.24%. Our NGS test was validated at a LLOD at 5%, and not applicable for MRD detection. However, the pathogenic mutations of RUNX1, ASXL1, EZH2 and FLT3 present in the initial diagnostic marrow were suitable to track for residual and persistent disease. Presumably, the monocytes also carried these mutations in the initial diagnostic bone marrow. We found that monocytes (about 5%, around the level of our NGS LLOD) lost the phenotypic aberrancies, i.e. no expression of CD2 and CD5 at the one-month follow up bone marrow when AML MRD by flow cytometry was performed. Monocytic markers employed in our AML MRD panel (CD14, CD36, CD64, and CD163) did not display phenotypic aberrancies in the monocytes. NGS on the same follow up bone marrow sample did not detect the pathogenic gene mutations that were present in the initial bone marrow. These findings may suggest negative AML MRD results.

As pointed out by Paietta et al. in 2018 [23], the ideal AML MRD assay for each clinical situation will most likely depend on the individual patient’s characteristics and the AML subtype. Our case illustrates that in pDC-AML, MFC and molecular study including NGS for MRD evaluation may be complementary. Even a NGS test without improved sensitivity or tailored for MRD detection might be useful.

Conclusions

pDC-AML is a newly described subtype of acute myeloid leukemia, with immunophenotypic and molecular features distinct from BPDCN and MPDCP associated with other myeloid neoplasms. Flow cytometry identification of immature markers on pDCs and continuous maturation with the myeloblasts are important in the differential diagnosis. AML MRD by flow cytometry for pDC-AML may require certain considerations, such as the phenotype of pDCs and mature monocytes, but a molecular approach including NGS might be of help in the final AML MRD determination. Clinical, pathologic, and molecular correlations are important for a final diagnosis of this rare entity.

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