ICCS Case of the Quarter 65 year old male with soft tissue mass in the thigh No significant past medical history Blood pressure medication only A biopsy of the soft tissue mass was performed, with subsequent bone marrow evaluation. CBC WBC 4.8 Hct 52.4 Plt 189

Bone marrow: flow cytometry

Antibodies and Fluorochromes PB* V450@ BV421# FITC PE PE-TR* PE -CF594# PE Cy5* PE Cy55# PE Cy7 A594 APC APC-A700 APC-H7 HLA-DR* CD15 CD33 CD19# CD117* CD13 CD38 CD34 CD71 CD45 CD64 CD123 CD4* CD14# CD16 CD20@ Kappa Lambda CD5# CD19 CD10 CD8# CD2 CD5 CD34# CD56* CD3 CD4 CD7 CD30 CD45@ cMPO cCD79a cCD3* Tubes M1 M2 B T Cytoplasmic

Starting Point 2. 1. 3. We start by obtaining single cell data on all events, avoiding cell clumps, by gating on “singlets” based on forward scatter height (FSC-H) versus area characteristics (FSC-A). Next we focus on the “singlet gate,” and gate based on FSC-A and side scatter height(SSC-H) characteristics in order to obtain viable events, and also exclude nucleated red cells. Now we have white cell data, and we gate on the viable cellular components based on CD45 versus SSC-H characteristics to divide into cell types. Blue arrow is the lymphocyte gate Green arrow is the myeloid and monocytic gate Red arrow is the blast or immature gate.

Next: Focus on the dim CD45 / blast gate (RED events) 1. 2. CD45 vs SCC defined Blast gate The events in the dim CD45 vs SCC defined blast region are highlighted in red, and negative for CD34 and CD19, Looking at all WBCs for expression of CD117, the RED, dim CD45 population is essentially negative for CD117

Myeloid tubes, M1 and M2 In the the myeloid tubes the dim CD45, Red population is: Positive for low CD4, CD33, CD38, subset low CD71, bright CD123, and HLA-DR Negative or essentially negative for CD15, CD16, CD19, CD34 and CD64. CD13, CD14 and CD117 may show small subset expression, but are overall negative

B and T cells tubes The B and T cell tubes show the dim CD45, Red population is positive for CD56 (intermediate to bright) with low CD4, low to intermediate CD7 and negative for CD2, CD3, CD5, CD8, CD10 and CD20

Cytoplasmic tube Phenotype by flow cytometry The cytoplasmic tube shows that the dim CD45, Red population is negative for cCD3, cCD79a and cMPO. There is spill over of maturing myeloid cells from the myeloid gate (blue arrow) into the dim CD45 gate, which is colored aqua in the dim CD45 gated dot plots. Phenotype by flow cytometry Positive: CD4 (low), CD7 (low to intermediate), CD13 (subset low), CD33, CD38, CD45 (dim), CD56 (intermediate to bright), CD71 (subset low), CD123 (bright) and HLA-DR Negative (or essentially negative): CD2, CD3 (surface and cytoplasmic), CD5, CD8, CD10, CD13, CD14, CD15, CD16, CD19, CD20, CD34, CD64, cCD79a, CD117, cMPO

Soft tissue mass (thigh): morphology

Soft tissue touch prep, Wright-Geimsa, 40x Numerous, monotonous large mononuclear cells -Moderate amount of cytoplasm, some with vacuoles, no granules -Nuclei round/oval to folded with smooth/delicate chromatin, some with nucleoli

Soft tissue touch prep, Wright Geimsa, 100x

H&E, 40x Diffuse sheets of intermediate to large mononuclear cells -Scant to moderate amount of cytoplasm -Nuclei round/oval to folded, some elongate, with smooth chromatin -Some with nucleoli Blue arrow points to reactive lymphocytes for comparison

H&E, 63x

Immunohistochemistry TCL-1 positive, 10x TDT positive, 10x

Diagnosis: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) Involving the skin and marrow Flow cytometry: positive CD4 (low), CD7 (low to intermediate), CD33, CD38, CD45 (dim), CD56 (intermediate to bright), CD71 (subset low), CD123 (bright) and HLA-DR Immunohistochemistry: positive for TCL-1 and TDT

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) Although lineage had previously been under debate, it is now classified with acute myeloid leukemia in the 2008 WHO classification. Synonyms / historical designations: Agranular CD4/CD56 positive hematodermic neoplasm, Blastic NK cell leukemia/lymphoma

BPDCN cell of origin Plasmacytoid dendritic cell (PDC) / plasmacytoid monocyte Small numbers are normally present in peripheral blood/marrow and lymphoid tissues (primary and secondary) with proliferations occurring in various disease states Such as Toxoplasmosis, Castleman’s and various hematopoietic and non-hematopoietic neoplasms Normal PDCs express CD123 and lack CD56 Although a minor population of normal CD56 positive PDC-like cells have been noted in the peripheral blood and marrow 0.03% of peripheral blood mononuclear cells

BPCDN Clinical Presentation Usually occurs in the the 6th decade (although can occur at any age, including pediatric cases). Typically presents with cutaneous lesions, which often include marrow involvement Fewer cases will lack dermal involvement, presenting with a leukemic picture Some cases will involve marrow and skin without blood involvement Cutaneous lesions may vary from small plaques and nodules to large plaques/tumors with variable pigmentation (tan/brown – red – blue/purple) May have a bruise-like appearance May be single or multiple

BPDCN morphology Blast or blast-like morphology Intermediate to large mononuclear cells with round/oval nuclei, may have nuclear irregularity or folding Scant to moderate amount of agranular cytoplasm, may have vacuoles (typically localize along cell membrane) Smooth/delicate/fine chromatin, often with nucleoli “Leukemic” type of infiltration in skin and lymph nodes Dermal infiltrate, perivascular and periadnexal which does not involve the epidermis. Progresses to diffuse sheets in the dermis / subcutaneous tissues. Early lymph node involvement of medullary and sinus regions progressing to infiltration of the cortical/paracortical regions. Diffuse sheets with progression.

BPDCN Immunophenotype Typical phenotype: expression of CD4, CD38, CD43, CD56, CD123 (typically brighter than monocytes), TCL1, CD303 (BDCA-2), CD2AP and HLA-DR CD7, CD33, CD68, TdT and S100 expression can be seen. lack of surface and cytoplasmic CD3, CD5, CD13, CD14, CD15, CD16, CD19, CD20, CD34, CD64, CD117, EBER, Lysozyme, MPO, NSE, TIA-1

BPDCN Immunophenotype The five most characteristic markers are CD4, CD56, CD123, CD303 and TCL1 Non typical phenotypic findings Although typically negative, low CD2 or CD13 may be seen Weak expression of CD4 or CD56 (and extremely rare lack of CD56 and/or CD4) has been reported If lacking CD56, exclusion of proliferating mature plasmacytoid dendritic cells must be excluded

BPDCN cytogenetics/molecular No specific cytogenetic or molecular alterations Often has a complex karyotype Frequent deletions of TP53, CDKN2A, CDKN1B, RB1, TET2, PTEN (tumor suppressor genes)

BPDCN Clinical Course Extremely poor prognosis in adults Usually responds to therapy, but quickly relapses Average overall survival 12-14 months Variable chemotherapeutic regimens have been used, including CHOP*, ALL type induction and AML type induction regimens Although most patients will respond initially to chemotherapy, many will relapse Best treatment protocol is not known; however, long term survival in adults appears best achieved with stem cell transplant in first remission. BPDCN is clinically less aggressive in children Typically treated with a high-risk ALL type regimen Stem cell transplant may be reserved for those who relapse in second remission *CHOP – cyclophosphamide, doxorubicin, vincristine, prednisone

Differential diagnosis NK cell neoplasm Acute Monocytic/Myeloid leukemia (AMoL/AML) Similar to BPDCN, NK cell neoplasms will express CD43 and CD56 Unlike BPDCN, NK cell neoplasms will lack CD4, CD123, TCL1, TdT Although CD7 can be expressed in both NK cell neoplasms and BPDCN, CD7 is typically brighter and more uniform in NK cell neoplasms NK cell neoplasms will express EBER and BPDCN will be negative Similar to BPDCN, AMoL will often express CD4, CD33 and HLA-DR, may express CD56 and typically lack CD14. Unlike BPDCN, AMoL lacks TCL1, CD303 (BDCA-2) and express bright CD64 and lysozyme. Although CD123 is expressed in both AMoL and BPDCN, CD123 is typically brighter in BPDCN. AML will often express CD34 and/or CD117, where BPDCN will be negative

References Swerdlow SH, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, IARC, Lyon, p145-47, 2008. Cherian S, Wood B. Flow Cytometry in evaluation of hematopoietic neoplasms: A case-based approach, CAP press, IL, p152-55, 2012. Laribi K, et al. “Blastic Plasmacytoid Dendritic Cell Neoplasm: From Origin of the cell to Targeted therapy; Biol Blood Marrow Transplant, 1-11, 2016. Yang S, Wang E. Blastic Plasmacytoid Dendritic Cell Neoplasm: A clinicopathologic review, Arch Pathol Lab Med; 138, 564-9, 2014. Julia F, et al. Blastic Plasmacytoid Dendritic Cell Neoplasms: Clinico-immunohistochemical Correlations in a Series of 91 Patients, Am J Surg Pathol; 38(5)673-80:2014. Riaz W et al. Blastic Plasmacytoid Dendritic Cell Neoplasm: Update on Molecular Biology, Diagnosis and Therapy, Cancer Control; 21(4)279-89:2014. Jegallan A, et al. Blastic plasmacytoid dendritic cell neoplasm in children: diagnostic features and clinical implications, haematologica, 95(11)1873-9: 2010. Osaki Y et al. Characterization of CD56+ dendritic-like cells: a normal counterpart of blastic plasmacytoid dendritic cells neoplasm?, PLoS One, 8(11)e8172:2013