Flow cytometry is used in cell-based diagnostic evaluation for blood-borne malignancies including leukemia and lymphoma. The current practice for cytometry data analysis relies on manual gating to identify cell subsets in complex mixtures, which is subjective, labor-intensive, and poorly reproducible. This article reviews recent efforts to develop, validate, and disseminate automated computational methods and pipelines for cytometry data analysis that could help overcome the limitations of manual analysis and provide for efficient and data-driven diagnostic applications. It demonstrates the performance of an optimized computational pipeline in a pilot study of chronic lymphocytic leukemia data from the authors' clinical diagnostic laboratory.

Targeting BTK has profoundly changed the face of CLL treatment over the past decade. Iterative advances in the cat and mouse game of resistance and redesign have moved BTK inhibitors from covalent to non-covalent and now targeted protein degraders. However, contrary to the presumption that protein degraders may be impervious to mutations in BTK, we now present clinical evidence that a mutation in the kinase domain of BTK, namely A428D, can confer disease resistance to a BTK degrader currently in clinical trials, that is BGB-16673. Modeling of a BTK A428D mutation places a negatively charged aspartic acid in place of the hydrophobic side chain of alanine within the binding pocket of another BTK-degrader in clinical development, namely NX-2127, suggesting that CLL cells with BTK A428D also may be resistant to NX-2127, as they already are known to be with either non-covalent or covalent inhibitors of BTK. Consequently, the two BTK degraders furthest advanced in clinical trials potentially may select for CLL cells with BTK A428D that are resistant to all approved BTKis.

Classical Hodgkin lymphoma (CHL) is recognized as a B-cell neoplasm arising from germinal center or postgerminal center B-cells. The hallmark of CHL is the presence of CD30 (+) Hodgkin and Reed-Sternberg (HRS) cells with dim expression of PAX5. Nearly all of the HRS cells are positive for PAX5. However, a small minority of HRS cells may lack PAX5 expression, which can cause a diagnostic dilemma. Herein we describe two cases of PAX5-negative CHL and review of the English literature on this very rare entity. It is crucial to be aware of this phenomenon, which in some cases may lead to misdiagnosis and may ultimately adversely affect patient's management.

BRAF mutations are associated with a small number of hematologic malignancies, including hairy cell leukemia and histiocytic disorders. In addition, BRAF mutations have also been detected in low frequency in other B-cell lymphomas, such as chronic lymphocytic leukemia and diffuse large B-cell lymphoma, but never in mantle cell lymphoma (MCL). We present a case of a 69-year-old female with classic MCL harboring a BRAFN581S mutation. To our knowledge, this is the first reported case of any BRAF mutation in MCL.

Erdheim-Chester disease (ECD) is a rare form of non-Langerhans cell histiocytosis characterized by xanthogranulomatous infiltration of foamy histiocytes frequently involving bone and other organ systems. We herein report a unique case of ECD discovered incidentally in an explanted liver in a 65-year-old male with end-stage liver disease secondary to hepatitis C cirrhosis. Histological examination and immunohistochemical studies in the explanted liver revealed prominent foamy histiocytes that were CD68 positive, but CD1a and S100 negative. Mutational hotspot analysis of the explanted liver using a panel of 47 most common cancer-related genes performed by next generation sequencing (NGS) revealed likely somatic mutations in the PDGFRA, PTEN, and HNF1A genes, but no BRAF codon 600 mutations were detected. The bone marrow showed similar findings as in the liver. Whole body PET and bone scans demonstrated increased heterogeneous uptake in bilateral humeral and femoral diaphysis, most compatible with ECD. To our knowledge, this is the first case report of ECD that involves mainly bone marrow and liver with novel genomic alterations. Our case highlights the diversity and complexity of this disease entity and the importance of multi-modality approach integrating clinical and radiologic features with histopathologic and molecular/genomic findings.

Pathogenesis of Richter transformation (RT) or Richter syndrome (RS) of chronic lymphocytic leukemia (CLL) is still largely unknown. Increasing evidences show that c-MYC may play a role in the development of RS. Here we report three cases of RS with overexpression of c-MYC. The first case was a 78-year-old male who initially presented with CLL and then developed diffuse lymphadenopathy and ascites shortly after. Ascites cytology showed a population of large lymphoid cells positive for MYC (8q24) rearrangement by fluorescence in situ hybridization (FISH) and overexpression of c-MYC by immunohistochemistry (IHC). The second case was a 66-year-old male presented with rapidly enlarging lymph nodes and pleural effusion after a long history of CLL. Biopsy showed large B-cells positive for c-MYC overexpression and high Ki-67 proliferation index (80-90%). The third case was a 62-year-old female with CLL who presented for lobectomy for lung adenocarcinoma. Interestingly, along with the carcinoma, large B-cell lymphoma was incidentally found which had the same immunophenotype as the CLL. FISH analysis revealed gain of c-MYC at 8q and IHC showed increased c-MYC expression. This study supports that c-MYC plays a critical role in RS.

A 52 year-old man with Erdheim-Chester Disease (ECD) (a non-Langerhans polyostotic sclerosing histiocytosis) had next-generation sequencing (NGS) performed as part of his diagnostic workup. In addition to the tissue BRAF V600E mutation that is found in over 50% of ECD cases, he was also found to have a JAK2 V617F alteration in cell-free circulating tumor DNA (ctDNA) (liquid biopsy). The latter was thought to be an "incidental" finding, perhaps due to clonal hematopoiesis (though this usually occurs in older individuals), as his blood counts were normal and he had no splenomegaly. Approximately 13 months after the ctDNA test showing JAK2 V617F, he developed anemia, thrombocytopenia, and splenomegaly. Marrow biopsy then showed megakaryocytic atypia and markedly increased marrow fibrosis, consistent with WHO grade 2 of 3 myelofibrosis. Therefore, the patient was determined to have ECD with a typical BRAF V600E mutation, as well as primary myelofibrosis, with the latter diagnosis manifesting clinically over one year after the JAK2 V617F was first detected in ctDNA. He recently was started on the JAK2 inhibitor ruxolitinib. This case demonstrates that genomic alterations detected by liquid biopsy for evaluation of specific malignancies already present may serve as an early harbinger of hematological disease.

Interdigitating dendritic cell sarcoma (IDCS) and histiocytic sarcoma (HS) are two distinct rare hematolymphoid neoplasms, and HS derived from a likely pre-existing IDCS has never been reported in the English literature. Diagnosis of such entities in excised specimens is difficult, but becomes more difficult with the scant amount of materials obtained with fine needle aspiration (FNA) and core needle biopsy. Here we present an interesting and unique case of an IDCS located within a mesenteric mass, which was initially diagnosed as IDCS from the cytology of FNA and core needle biopsy specimens. After brief chemotherapy, the patient again developed abdominal pain, and a HS was diagnosed based on the excised segmental small intestinal specimen. While the exact relationship between the IDCS and HS cannot be ascertained, it is most likely that the HS is derived from the IDCS, although co-existing HS in addition to IDCS from the cytology specimen cannot be completely ruled out.

Near early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a rare hematologic malignancy, for which second line therapeutic options are limited. T-cell leukemias are also rarely associated with leukemia cutis, which is more often seen in leukemias of myeloid origin. We present the case of an adult male diagnosed with near ETP-ALL, with IDH2 and DNMT3A mutations, suggestive of a myeloid origin, and leukemia cutis. After the patient progressed on hyper-CVAD and nelarabine, we treated him with the BCL-2 inhibitor venetoclax and the hypomethylating agent decitabine. The regimen induced a rapid bone marrow response and resolution of the leukemia cutis.