Consensus opinion from an international group of experts on measurable residual disease in hairy cell leukemia.

A significant body of literature has been generated related to the detection of measurable residual disease (MRD) at the time of achieving complete remission (CR) in patients with hairy cell leukemia (HCL). However, due to the indolent nature of the disease as well as reports suggesting long-term survival in patients treated with a single course of a nucleoside analog albeit without evidence of cure, the merits of detection of MRD and attempts to eradicate it have been debated. Studies utilizing novel strategies in the relapse setting have demonstrated the utility of achieving CR with undetectable MRD (uMRD) in prolonging the duration of remission. Several assays including immunohistochemical analysis of bone marrow specimens, multi-parameter flow cytometry and molecular assays to detect the mutant BRAF V600E gene or the consensus primer for the immunoglobulin heavy chain gene (IGH) rearrangement have been utilized with few comparative studies. Here we provide a consensus report on the available data, the potential merits of MRD assessment in the front-line and relapse settings and recommendations on future role of MRD assessment in HCL.

Development of a distributed international patient data registry for hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare lymphoproliferative disorder, comprising only 2% of all leukemias. The Hairy Cell Leukemia Foundation (HCLF) has developed a patient data registry to enable investigators to better study the clinical features, treatment outcomes, and complications of patients with HCL. This system utilizes a centralized registry architecture. Patients are enrolled at HCL Centers of Excellence (COE) or via a web-based portal. All data are de-identified, which reduces regulatory burden and increases opportunities for data access and re-use. To date, 579 patients have been enrolled in the registry. Efforts are underway to engage additional COE's to expand access to patients across the globe. This international PDR will enable researchers to study outcomes in HCL in ways not previously possible due to the rarity of the disease and will serve as a platform for future prospective research.

Validation, Implementation, and Clinical Impact of the Oncomine Myeloid Targeted-Amplicon DNA and RNA Ion Semiconductor Sequencing Assay.

The identification of clinically significant genes recurrently mutated in myeloid malignancies necessitates expanding diagnostic testing with higher throughput, such as targeted next-generation sequencing. We present validation of the Thermo Fisher Oncomine Myeloid Next-Generation Sequencing Panel (OMP), targeting 40 genes and 29 fusion drivers recurrently mutated in myeloid malignancies. The study includes data from a sample exchange between two Canadian hospitals demonstrating high concordance for detection of DNA and RNA aberrations. Clinical validation demonstrates high accuracy, sensitivity, and specificity of the OMP, with a lower limit of detection of 5% for single-nucleotide variants and 10% for insertions/deletions. Prospective sequencing was performed for 187 samples from 168 unique patients presenting with suspected or confirmed myeloid malignancy and other hematological conditions to assess clinical impact of identifying variants. Of detected variants, 48% facilitated or clarified diagnoses, 29% affected prognoses, and 25% had the potential to influence clinical management. Of note, OMP was essential to identifying patients with premalignant clonal states likely contributing to cytopenias. We also found that the detection of even a single variant by the OMP assay, versus 0 variants, was predictive of overall survival, independent of age, sex, or diagnosis (P = 0.03). This study demonstrates that molecular profiling of myeloid malignancies with the OMP represents a promising strategy to advance molecular diagnostics.

Multicenter observational study evaluating the impact of platelet transport bags on product wastage.

BACKGROUND: Platelets are the most commonly discarded blood product in Canada, with the most common cause of in-date product loss being improper storage. Transport containers to maintain temperature and extend acceptable return time may represent a method to reduce wastage. The objective of this study was to evaluate the impact of a validated Platelet Transport Bag (PTB) on platelet wastage. STUDY DESIGN AND METHODS: Thirty-six hospitals with the highest platelet discards were invited to participate in a before-after observational study. Hospitals were instructed to utilize a validated 4-h PTB for clinical situations where immediate transfusion was not planned. Five hospitals audited in-date platelet discards from July 2018 to November 2019 to characterize wastage causes. In-date platelet discard data 12 months before and after the start date for each site were analyzed to determine changes in wastage. RESULTS: Of 36 hospital sites, 16 agreed to participate. Pre- and postdiscards were 277 and 301, respectively, for all sites combined. There were no significant before-after change in wastage rate (+0.05%, p = .51). Fifty discards were included in the detailed audit; the most common reasons were return to the blood bank after more than 60 min outside a PTB (n = 17, 34%) and return in a red cell cooler (n = 10, 20%). CONCLUSION: Implementation of PTB did not improve wastage. Common causes of in-date discards were return after 1 h outside of a PTB and placement in a red cell cooler in error. Further research is required to investigate potential strategies to mitigate in-date platelet wastage.

PD-L1 lineage-specific quantification in malignant pleural effusions of lung adenocarcinoma by flow cytometry.

OBJECTIVES: Pathologists encounter several challenges with programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) tests in malignant effusions, including lineage specification (distinction between carcinoma vs. immune and mesothelial cells), background staining, sample fixation issues and inter-observer variability. We explored flow cytometric (FC) quantification of PD-L1 expression in malignant pleural effusions of lung adenocarcinoma patients as an alternative, automated, and objective quantification method compared to PD-L1 IHC. MATERIALS AND METHODS: We examined 23 malignant pleural effusions of TTF-1-positive adenocarcinoma were subjected to FC with a panel of antibodies against CD45, CD3, CD200, EpCAM, D2-40 (podoplanin), and PD-L1 (clone MIH1). The PD-L1 gate was established using fluorescence-minus-one (FMO) isotype controls. Lineage-specific PD-L1 surface expression was quantified and the FC tumor proportion score (TPS) was assessed. PD-L1 IHC was performed on cell block sections using Dako PD-L1 IHC 22C3 pharmDx assay and assessed by two cytopathologists blinded to the FC PD-L1 TPS. RESULTS: FC analysis allowed for the distinction between carcinoma cells (CD45-/EpCAM+/D2-40-), leukocytes (CD45+/EpCAM-/D2-40-) and mesothelial cells (CD45-/EpCAM-/D2-40+). FC PD-L1 TPS ranged from 0% to 77 %, while the 22C3 IHC PD-L1 TPS ranged from 0% to 97 %. The FC and IHC TPS values correlated positively (R = 0.8). Best concordance was observed when FC was performed and cell blocks were generated in parallel (R = 0.99). FC also allowed for simultaneous PD-L1 quantification in mesothelial and T-cells. PD-L1 expression on mesothelial cells ranged from 0% to 90.9 %, which also correlated positively with IHC TPS (R = 0.54). PD-L1 expression on T-cells was limited (0.1-2.9 %). CONCLUSION: FC permits rapid, objective and lineage-specific PD-L1 surface expression quantification with limited specimen manipulation. The FC and IHC concordance was impacted by different antibody clones being used, but the positive correlation suggests potential clinical utility, especially in malignant effusion specimens.

High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms.

BACKGROUND: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by an inability to make Glyco-Phosphatidyl-Inositol (GPI)-linked cell surface structures. Fluorescent proaerolysin (FLAER-Alexa488) is increasingly used to detect GPI-deficient WBCs by flow cytometry. However, FLAER is not available in all countries and is expensive to obtain in others. An earlier study to compare FLAER-based and non-FLAER assays confirmed very good agreement between the two tubes suggesting a cost effective simultaneous evaluation of PNH neutrophils and monocytes is possible without FLAER. METHODS: We have used a single tube approach with a 7-color assay comprising FLAER-CD157-CD15-CD64-CD24-CD14-CD45. Conjugates were carefully selected and validated so that stained samples could be analyzed on either 10-color Navios or 8-color FACSCanto II platforms. The 6-color (minus CD14) and 5-color (minus CD24 and CD14) versions were also developed and compared with our predicate clinical lab 5-color assay comprising FLAER-CD157PE-CD64ECD-CD15PC5-CD45PC7. RESULTS/CONCLUSIONS: CD15-gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64-gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER-based plots derive virtually identical data to the non-FLAER plot for neutrophils (R2 = 1) and monocytes (R2 = 0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7-, 6-, and 5-color versions of the assay. Assessment of non-PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches. © 2018 International Clinical Cytometry Society.

Consensus guidelines for the diagnosis and management of patients with classic hairy cell leukemia.

Hairy cell leukemia is an uncommon hematologic malignancy characterized by pancytopenia and marked susceptibility to infection. Tremendous progress in the management of patients with this disease has resulted in high response rates and improved survival, yet relapse and an appropriate approach to re-treatment present continuing areas for research. The disease and its effective treatment are associated with immunosuppression. Because more patients are being treated with alternative programs, comparison of results will require general agreement on definitions of response, relapse, and methods of determining minimal residual disease. The development of internationally accepted, reproducible criteria is of paramount importance in evaluating and comparing clinical trials to provide optimal care. Despite the success achieved in managing these patients, continued participation in available clinical trials in the first-line and particularly in the relapse setting is highly recommended. The Hairy Cell Leukemia Foundation convened an international conference to provide common definitions and structure to guide current management. There is substantial opportunity for continued research in this disease. In addition to the importance of optimizing the prevention and management of the serious risk of infection, organized evaluations of minimal residual disease and treatment at relapse offer ample opportunities for clinical research. Finally, a scholarly evaluation of quality of life in the increasing number of survivors of this now manageable chronic illness merits further study. The development of consensus guidelines for this disease offers a framework for continued enhancement of the outcome for patients.

Incremental value of the bone marrow trephine biopsy in detecting residual leukemia following treatment for Acute Myeloid Leukemia.

Most guidelines suggest that only the bone marrow aspirate (BMA) is necessary to assess residual disease following intensive chemotherapy for Acute Myeloid Leukemia (AML) with the bone marrow trephine biopsy (BMTB) recommended in cases of a poor quality BMA. We performed a retrospective study evaluating this in a cohort of patients receiving intensive chemotherapy for AML. Residual disease was assessed by morphological examination of the BMA and BMTB±immunohistochemistry. Of the 647 marrows 32.6% were interim marrows performed prior to peripheral count recovery, 41.7% were end of induction (EOI) marrows and the remaining were 'other marrows'. The BMA and BMTB findings were concordant in 92.8% of cases. The BMTB led to a change in diagnosis from 'no leukemia' to 'residual leukemia' in 5.2% of interim, 3.7% of EOI and 2.4% of 'other' marrows. The BMA alone had a sensitivity of 86.8% in detecting residual leukemia and of 82.3%, 82.5% and 94.2% for interim, EOI and 'other marrows', respectively. Despite the high concordance between the BMA and the BMTB the poor sensitivity of the BMA in detecting residual leukemia, particularly at EOI, may lead to an overestimation of the complete remission rates which may have therapeutic and clinical trial implications.

Clinical features and diagnosis of hairy cell leukemia.

Significant advances in the diagnosis and treatment of hairy cell leukemia (HCL) have recently been made. Improved distinction of HCL from its mimics though clinical presentations, morphologic and immunophenotypic features, and more recently molecular biology, has highlighted marked differences in treatment response and overall prognosis between these disorders. As our understanding of the unique pathobiology of HCL has grown, exciting new avenues of treatment as well as insight into immune function have been obtained. This review provides an overview of the clinical features and diagnostic attributes of HCL, with contrast to other mature B cell lymphoproliferative disorders with overlapping features.

Transfusion-related acute lung injury after transfusion of pooled immune globulin: a case report.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a severe transfusion reaction that manifests as acute respiratory compromise within 6 hours of the infusion of blood products. Intravenous immune globulin (IVIG) is prepared from large pools of human plasma and is commonly administered in the outpatient setting for the treatment of a wide range of diseases. As a plasma-derived blood product, IVIG may also cause TRALI, although reports of this are exceedingly rare. CASE REPORT: A 77-year-old female with common variable immune deficiency had been receiving IVIG since 1996 for infection prophylaxis. During a scheduled infusion, the patient developed hypertension and dyspnea, requiring increasing oxygen supplementation and subsequent intubation. Radiographic studies demonstrated the bilateral chest infiltrates, with no evidence of infection or circulatory overload. The patient was extubated after 24 hours and discharged several days later. The patient had not previously received this lot of IVIG and has since received further transfusions with different lot numbers of the same product without incident. CONCLUSION: This case report documents a case of TRALI after IVIG transfusion. While a very rare cause, this case furthers evidence that TRALI can occur after IVIG transfusion.

Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag.

BACKGROUND: The identification and characterization of several interferon (IFN)-induced cellular HIV-1 restriction factors, defined as host cellular proteins or factors that restrict or inhibit the HIV-1 life cycle, have provided insight into the IFN response towards HIV-1 infection and identified new therapeutic targets for HIV-1 infection. To further characterize the mechanism underlying restriction of the late stages of HIV-1 replication, we assessed the ability of IFNbeta-induced genes to restrict HIV-1 Gag particle production and have identified a potentially novel host factor called HECT domain and RCC1-like domain-containing protein 5 (HERC5) that blocks a unique late stage of the HIV-1 life cycle. RESULTS: HERC5 inhibited the replication of HIV-1 over multiple rounds of infection and was found to target a late stage of HIV-1 particle production. The E3 ligase activity of HERC5 was required for blocking HIV-1 Gag particle production and correlated with the post-translational modification of Gag with ISG15. HERC5 interacted with HIV-1 Gag and did not alter trafficking of HIV-1 Gag to the plasma membrane. Electron microscopy revealed that the assembly of HIV-1 Gag particles was arrested at the plasma membrane, at an early stage of assembly. The mechanism of HERC5-induced restriction of HIV-1 particle production is distinct from the mechanism underlying HIV-1 restriction by the expression of ISG15 alone, which acts at a later step in particle release. Moreover, HERC5 restricted murine leukemia virus (MLV) Gag particle production, showing that HERC5 is effective in restricting Gag particle production of an evolutionarily divergent retrovirus. CONCLUSIONS: HERC5 represents a potential new host factor that blocks an early stage of retroviral Gag particle assembly. With no apparent HIV-1 protein that directly counteracts it, HERC5 may represent a new candidate for HIV/AIDS therapy.