Immunohistochemistry screening for TP53 mutation in myeloid neoplasms in AZF-fixed bone marrow biopsies.

TP53 mutational status in myeloid neoplasms is prognostic and in acute myeloid leukaemia (AML) may lead to alternative induction therapy; therefore, rapid assessment is necessary for precision treatment. Assessment of multiple prognostic genes by next generation sequencing in AML is standard of care, but the turn-around time often cannot support rapid clinical decision making. Studies in haematological neoplasms suggest p53 immunohistochemistry (IHC) correlates with TP53 mutational status, but they have used variable criteria to define TP53 overexpression. p53 IHC was performed and interpreted on AZF-fixed, acid decalcified bone marrow biopsies on 47 cases of clonal myeloid neoplasms with TP53 mutations between 2016 and 2019 and 16 control samples. Results were scored by manual and digital analysis. Most TP53-mutated cases (81%) overexpressed p53 by digital analysis and manual analysis gave similar results. Among the nine TP53-mutated IHC-negative cases, seven (78%) were truncating mutations and two (22%) were single-hit missense mutations. Using a digital cut-off of at least 3% ≥1+ positive nuclei, the sensitivity and specificity are 81% and 100%; cases with loss-of-function mutations were more likely to be negative. In this cohort, p53 immunopositivity correlated with TP53 mutational status, especially missense mutations, with excellent specificity. Truncating TP53 mutations explain most IHC-negative cases, impacting the sensitivity. We demonstrate that p53 IHC can screen for TP53 mutations allowing quicker treatment decisions for most patients. However, not all patients will be identified, so molecular studies are required. Furthermore, cut-offs for positivity vary in the literature, consequently laboratories should independently validate their processes before adopting p53 IHC for clinical use. p53 IHC performs well to screen for TP53 mutations in AZF-fixed bone marrow. Performance in our setting differs from the literature, which shows variability of pre-analytic factors and cut-offs used to screen for TP53 mutations. Each laboratory should validate p53 IHC to screen for TP53 mutations in their unique setting.

High-grade B-cell lymphoma, not otherwise specified: a multi-institutional retrospective study.

In this multi-institutional retrospective study, we examined the characteristics and outcomes of 160 patients with high-grade B-cell lymphoma, not otherwise specified (HGBL-NOS)-a rare category defined by high-grade morphologic features and lack of MYC rearrangements with BCL2 and/or BCL6 rearrangements ("double hit"). Our results show that HGBL-NOS tumors are heterogeneous: 83% of patients had a germinal center B-cell immunophenotype, 37% a dual-expressor immunophenotype (MYC and BCL2 expression), 28% MYC rearrangement, 13% BCL2 rearrangement, and 11% BCL6 rearrangement. Most patients presented with stage IV disease, a high serum lactate dehydrogenase, and other high-risk clinical factors. Most frequent first-line regimens included dose-adjusted cyclophosphamide, doxorubicin, vincristine, and etoposide, with rituximab and prednisone (DA-EPOCH-R; 43%); rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP; 33%); or other intensive chemotherapy programs. We found no significant differences in the rates of complete response (CR), progression-free survival (PFS), or overall survival (OS) between these chemotherapy regimens. CR was attained by 69% of patients. PFS at 2 years was 55.2% and OS was 68.1%. In a multivariable model, the main prognostic factors for PFS and OS were poor performance status, lactate dehydrogenase >3 × upper limit of normal, and a dual-expressor immunophenotype. Age >60 years or presence of MYC rearrangement were not prognostic, but patients with TP53 alterations had a dismal PFS. Presence of MYC rearrangement was not predictive of better PFS in patients treated with DA-EPOCH-R vs R-CHOP. Improvements in the diagnostic criteria and therapeutic approaches beyond dose-intense chemotherapy are needed to overcome the unfavorable prognosis of patients with HGBL-NOS.

CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

A case of VEXAS syndrome associated with EBV-associated hemophagocytic lymphohistiocytosis.

Vacuoles, E1, X-linked, autoimmunity, somatic (VEXAS) syndrome is characterized by a pathogenic mutation in UBA1, which leads to protean complications including autoimmunity and myelodysplasia. A 56-year-old man with steroid-dependent, later steroid-refractory cutaneous polyarteritis nodosa and Sweet syndrome developed recurrent daily fever, macrocytic anemia, thrombocytopenia, acute hypoxic respiratory failure, and anasarca. He was eventually diagnosed with Epstein-Barr virus (EBV) viremia and hemophagocytic lymphohistiocytosis (HLH). He improved clinically with rituximab, ruxolitinib, and increased glucocorticoids before expiring from Pseudomonas sepsis. UBA1 exon 3 mutational analysis in myeloid enriched peripheral blood revealed a c.122T>C (p.Met41Thr) pathogenic variant, consistent with VEXAS syndrome. We describe the first case of EBV-associated HLH in a patient diagnosed with VEXAS syndrome. Early identification of this syndrome will be important in order to offer potential therapies before life-threatening complications arise.

Update on B-cell lymphoproliferative disorders of the gastrointestinal tract.

The gastrointestinal (GI) tract is home to a significant portion of the immune system, which interacts daily with the antigenic milieu of its contents. Therefore, the presence of white blood cells within the walls of the GI tract upon histologic examination is a familiar sight on GI biopsies-both in health and disease. The GI tract is the most common site of extranodal lymphomas, most of which are B-cell neoplasms. Here, we review common and uncommon B-cell neoplasms of the GI tract - extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), mantle cell lymphoma, duodenal-type follicular lymphoma, diffuse large B-cell lymphoma, plasmablastic lymphoma, EBV-positive mucocutaneous ulcer, and post-transplant lymphoproliferative disorders - with special focus on literature published during the past five years. Along with the other articles in this edition of Seminars in Diagnostic Pathology, it is the authors' hope that this review proves to be a useful resource in the workup of the array of hematopoietic processes that can involve the GI tract.

Clinician-ordered peripheral blood smears have low reimbursement and variable clinical value: a three-institution study, with suggestions for operational efficiency.

BACKGROUND: Peripheral blood smears are performed to evaluate a variety of hematologic and non-hematologic disorders. At the authors' institutions, clinician requests for pathologist-performed blood smear reviews have increased in recent years. Blood smears may contribute significantly to pathologists' workloads, yet their clinical value is variable, and professional reimbursement rates are low. This study aimed to identify clinical scenarios in which smear review is likely to provide value beyond automated laboratory testing. METHODS: Blood smear review practices at three institutions were examined, and the indications for and interpretations of clinician-initiated smears were reviewed to determine the percentage of smears with potential added clinical value. A smear review was classified as having added clinical value if the pathologist's interpretation included a morphologic abnormality that had the potential to impact patient management, and that could not be diagnosed by automated complete blood count with white blood cell differential or automated iron studies alone. RESULTS: Among 515 consecutive clinician-requested smears performed during the study timeframes, 23% yielded interpretations with potential added clinical value. When sorted by indication, 25, 19, and 13% of smear reviews requested for white blood cell abnormalities, red blood cell abnormalities, and platelet abnormalities, respectively, had findings with potential added clinical value. The proportion of smears with potential clinical value differed significantly across these three categories (p = 0.0375). CONCLUSIONS: Smear review ordering practices across three institutions resulted in a minority of smears with potential added clinical value. The likelihood of value varied according to the indication for which the smear was requested. Given this, efforts to improve the utilization and efficiency of smear review are worthwhile. Solutions are discussed, including engaging laboratory staff, educating clinicians, and modifying technology systems.

Prospective Investigation of an Automated PCR/Nucleic Acid Microarray-Based Platform for Enteric Pathogen Testing.

BACKGROUND: The Verigene Enteric Pathogens Test (Luminex Corporation) is a polymerase chain reaction (PCR)/nucleic acid microarray-based assay targeting 8 bacterial and viral pathogens that cause diarrhea. OBJECTIVE: To compare traditional enteric culture methods with stool testing by Verigene EP (PCR/microarray). METHODS: Tests were performed using PCR/microarray between February and August 2016. All specimens also underwent culture for Salmonella and Shigella; specimens that tested positive for bacterial pathogen(s) had confirmatory cultures. RESULTS: Valid results were obtained for 99.3% of the 3795 stool specimens. Among these, 497 (13.2%) specimens tested positive for at least 1 pathogen by PCR/microarray; 45.5% of these tested positive for 1 or more bacterial pathogens. Agreement between positive bacterial PCR/microarray results and culture-based testing was 85.3%. Compared with cultures, PCR/microarray demonstrated 95.2% and 87.5% sensitivity and 99.8% and 99.8% specificity for Salmonella and Shigella, respectively. CONCLUSIONS: The Verigene EP generated evaluable results for most stool specimens tested and demonstrated good agreement with bacterial cultures.