Novel ELANE Mutation Associated with a Clinical Presentation of Cyclic Neutropenia.

OBJECTIVES: Congenital neutropenia, defined by absolute neutrophil count (ANC) 2.5x109/L in infants, includes a variety of genotypic alterations that manifest with chronic immunodeficiency and, as a result, presents in infancy with recurrent infections. The gene that encodes neutrophil elastase, ELANE, has pathological variants yielding two distinct phenotypes: severe congenital neutropenia (SCN) and cyclic neutropenia (CyN). While SCN exhibits persistent pathologic ANC values, CyN exhibits pathologic ANC values in a patterned fashion which can recur in 21-day intervals. Here, we describe a patient with a novel heterozygous ELANE deletion (c.224+(4_19)del16) presenting with clinical features consistent with CyN and a response to first-line therapy of granulocyte colony- stimulating factor.

PCNEO, a New Proficiency Testing Program for Flow Cytometric Analysis of Plasma Cell Neoplasms From the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee.

CONTEXT.­: In 2018 the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee designed and implemented a new plasma cell neoplasia flow cytometry proficiency testing program-PCNEO-to allow clinical flow cytometry laboratories to monitor and assess their performance compared with a peer group. OBJECTIVE.­: To report the results from the first 4 years of the PCNEO program. DESIGN.­: Program participants were sent 2 sets of challenges per year, each including 1 wet challenge and 2 dry challenges, with associated clinical and laboratory findings. The wet challenges were composed of myeloma cell line specimens (with or without dilution in preserved whole blood) for flow cytometric analysis. The dry (paper) challenges were composed of clinical case summaries and images of flow cytometric test results from various flow cytometry laboratories of committee members. RESULTS.­: A total of 116 to 145 laboratories from 17 countries enrolled in the proficiency testing program. For the wet challenges, almost all participants (97%-100%; cumulative, 98.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of undiluted myeloma cell lines. Slightly fewer participants (89.0%-97.4%; cumulative, 95.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of diluted myeloma cell lines (10% or 50% dilutions into peripheral blood) intended to better represent a typical clinical sample. There was generally agreement among 80% or more of participants for positive or negative staining for CD38, CD138, CD19, CD20, and surface and cytoplasmic κ and λ light chains. Similarly, 84% to 100% of participants were able to correctly identify the presence of neoplastic plasma cell populations in paper challenges, including the presence of small, neoplastic plasma cell populations (0.01%-5.0% clonal plasma cells), or the presence of nonneoplastic plasma cell populations (correctly identified by 91%-96% of participants). CONCLUSIONS.­: Participant performance in the new proficiency testing program was excellent overall, with the vast majority of participants able to perform flow cytometric analysis and identify neoplastic plasma cell populations, and to identify small plasma cell clones or expanded populations of reactive plasma cells in dry challenge flow cytometry results. This program will allow laboratories to verify the accuracy of their testing program and test interpretations for the assessment of patients suspected of having a plasma cell neoplasm.

Summary of validation considerations with real-life examples using both qualitative and semiquantitative flow cytometry assays.

In the clinical laboratory, flow cytometry assays are critical to providing diagnostic and prognostic information to the treating clinicians. A validation or verification provides confidence that the assay will yield reliable results that can be trusted to make critical medical decisions. The following performance specifications should be included in a validation for laboratory developed tests as needed: accuracy (or trueness), precision (reproducibility and repeatability), detection capability, selectivity, reference range, and sample and reagent stability. We define these terms and present our approach to validation of several common flow cytometry assays, including examples of a leukemia/lymphoma assay and a paroxysmal nocturnal hemoglobinuria (PNH) assay.

Myelodysplastic Syndrome with Excess Blasts 2 (MDS-EB-2): A Historical Overview and Review of Forthcoming Classifications.

OBJECTIVES: Myelodysplastic syndrome with excess blasts-2 (MDS-EB-2) primarily affects adults older than 50 years and confers a worse prognosis with a higher risk of transformation to acute myeloid leukemia (AML) compared to myelodysplastic syndrome (MDS) and MDS with excess blasts-1 (MDS-EB-1). In ordering diagnostic studies for MDS, cytogenetic and genomic studies are vital as they have significant clinical and prognostic implications for the patient. We present a case of a 71-year-old male diagnosed with MDS-EB-2 with a pathogenic loss-of-function TP53 variant and discuss presentation, pathogenesis, and the importance of thorough diagnostic testing through multiple modalities to accurately diagnose and subtype MDS. In addition, we explore a historical overview of the diagnostic criteria of MDS-EB-2 and how it has changed over time from the World Health Organization (WHO) 4th edition in 2008, the WHO revised 4th edition in 2017, and the upcoming WHO 5th edition and International Consensus Classification (ICC) for 2022.

Multiple Myeloma Detected by Noninvasive Prenatal Testing.

OBJECTIVES: Noninvasive prenatal testing (NIPT) has become a widely used screening method to detect fetal aneuploidies using cell-free fetal DNA (cffDNA) obtained from maternal blood. It is noninvasive, highly sensitive and specific, and can be offered in the first trimester of pregnancy. Though the goal of NIPT is to detect abnormalities in fetal DNA, occasionally abnormalities are detected that are not attributable to the fetus. Tumor DNA is laden with abnormalities and, rarely, NIPT has detected occult malignancy in the mother. Maternal malignancy in pregnancy is relatively uncommon, estimated at 1 in 1,000 pregnant women. We present a case of a 38-year-old woman diagnosed with multiple myeloma after abnormal NIPT testing results.

Noninvasive Prenatal Testing Leading to a Diagnosis of Hodgkin Lymphoma.

OBJECTIVES: Noninvasive prenatal testing (NIPT) is a screening method used to detect the most common fetal aneuploidies using cell-free fetal DNA (cffDNA) obtained from maternal blood. Due to the high sensitivity and specificity, low false positive rate, and use as early as 10-weeks' gestation NIPT has been rapidly integrated into prenatal care. While NIPT is an excellent screening tool, the results can be influenced by many factors including placental mosaicism, maternal aneuploidy or mosaicism, and occult maternal malignancy. The diagnosis and treatment of malignancy during pregnancy present many challenges ranging from the use of imaging techniques to the delivery of optimal therapy, weighing the unique risks to both the mother and the fetus. We present a case of a 30-year-old woman diagnosed with Hodgkin lymphoma after NIPT and outline the challenges in diagnosis and treatment of malignancy occurring during pregnancy.

Mantle Cell Lymphoma Presenting with Cutaneous Lesions: A Rare Manifestation of a Systemic Disease.

OBJECTIVES: Cutaneous lymphoma is a broad term used to describe any type of lymphoma involving the skin. They may be primary, arising in the skin, or secondary, resulting from spread of a systemic lymphoma. Cutaneous involvement of mantle cell lymphoma (MCL) is extremely rare and most often occurs secondarily. To date, less than 100 cases of MCL involving the skin have been described in the English literature. We describe a case of MCL involving the skin as the clinical presentation of disease in a 74-year-old man and highlight the radiographic and pathologic findings, treatment course, and prognosis.

Acute Myeloid Leukemia with Myelodysplasia-Related Changes Presenting as Vitamin B12 Deficiency: A Cautionary Tale.

OBJECTIVES: Acute myeloid leukemia may present with significant dysmyelopoiesis within the peripheral blood smear and bone marrow aspirate. In the setting of Vitamin B12 deficiency, proliferation of a clonal population of malignant cells can become impaired, masking an underlying myelodysplastic or leukemic process. Typically, the cautionary tale warns against diagnosing acute myeloid leukemia before ruling out Vitamin B12 deficiency. Here we describe a patient who initially presented with pancytopenia and Vitamin B12 deficiency who, upon supplementation, developed overt acute myeloid leukemia. This case will highlight the importance of cytogenetic and molecular studies as essential diagnostic tools in patients with unique presentations.

Mantle cell lymphoma presenting with lethal atraumatic splenic rupture.

Mantle cell lymphoma is characterized by t(11;14) with CCND1-IGH fusion and manifests with a spectrum of disease ranging from relatively indolent to aggressive. Here, we present a case of pleomorphic mantle cell lymphoma with three fusion signals that presented with lethal atraumatic splenic rupture. We discuss on the implications of variant CCND1 signal patterns as well as the epidemiology and pathophysiology of atraumatic splenic rupture.

Mantle cell lymphoma presenting with lethal atraumatic splenic rupture

Mantle cell lymphoma is characterized by t(11;14) with CCND1-IGH fusion and manifests with a spectrum of disease ranging from relatively indolent to aggressive. Here, we present a case of pleomorphic mantle cell lymphoma with three fusion signals that presented with lethal atraumatic splenic rupture. We discuss on the implications of variant CCND1 signal patterns as well as the epidemiology and pathophysiology of atraumatic splenic rupture.

Educational Case: Cytogenetics.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.1.

Educational Case: Chronic Myeloid Leukemia.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.1.

High-grade B-Cell lymphoma with MYC and BCL6 rearrangements associated with Richter transformation of chronic lymphocytic leukemia

Richter transformation (RT), or Richter syndrome, is defined as the transformation of chronic lymphocytic leukemia (CLL) to an aggressive B-cell lymphoma. The vast majority, up to 99%, transform into diffuse large B-cell lymphoma (DLBCL), with a small subset (<1%) becoming classical Hodgkin lymphoma. Approximately half of RT cases progress through a pathway involving dysregulation of C-MYC. High-grade B-cell lymphoma (HGBL) is a recent diagnostic category of aggressive B-cell lymphomas set forth in the updated 2017 WHO Classification of Hematopoietic and Lymphoid Tissues. HGBL with MYC and BCL2 and/or BCL6 rearrangements, formerly "double-hit" and "triple-hit" lymphomas, comprise the majority of HGBL cases. Patients with HGBL have a worse prognosis than those with diffuse large B-cell lymphoma. We present a case of RT with rearrangements of MYC and BCL6. To our knowledge, there are no reported cases of RT with a "double-hit" lymphoma genotype.

High-grade B-Cell lymphoma with MYC and BCL6 rearrangements associated with Richter transformation of chronic lymphocytic leukemia.

Richter transformation (RT), or Richter syndrome, is defined as the transformation of chronic lymphocytic leukemia (CLL) to an aggressive B-cell lymphoma. The vast majority, up to 99%, transform into diffuse large B-cell lymphoma (DLBCL), with a small subset (<1%) becoming classical Hodgkin lymphoma. Approximately half of RT cases progress through a pathway involving dysregulation of C-MYC. High-grade B-cell lymphoma (HGBL) is a recent diagnostic category of aggressive B-cell lymphomas set forth in the updated 2017 WHO Classification of Hematopoietic and Lymphoid Tissues. HGBL with MYC and BCL2 and/or BCL6 rearrangements, formerly "double-hit" and "triple-hit" lymphomas, comprise the majority of HGBL cases. Patients with HGBL have a worse prognosis than those with diffuse large B-cell lymphoma. We present a case of RT with rearrangements of MYC and BCL6. To our knowledge, there are no reported cases of RT with a "double-hit" lymphoma genotype.

Trilineage Dysplasia in an Adolescent With Germline GATA2 Mutation.

The GATA family of DNA binding proteins consists of six different transcription factors (GATA1-6), each with diverse biologic function. The GATA2 protein has been shown to be vital for proliferation and maintenance of hematopoietic stem cells; mutations result in variable phenotypes including myelodysplastic syndrome.

Quantitative pixel intensity- and color-based image analysis on minimally compressed files: implications for whole-slide imaging.

Current best practice in the quantitative analysis of microscopy images dictates that image files should be saved in a lossless format such as TIFF. Use of lossy files, including those processed with the JPEG algorithm, is highly discouraged due to effects of compression on pixel characteristics. However, with the growing popularity of whole-slide imaging (WSI) and its attendant large file sizes, compressed image files are becoming more prevelent. This prompted us to perform a color-based quantitative pixel analysis of minimally compressed WSI images. Sections from three tissues stained with one of three reagents representing the colors blue (hematoxylin), red (Oil-Red-O), and brown (immunoperoxidase) were scanned with a whole slide imager in triplicate at 20x, 40x, and 63x magnifications. The resulting files were in the form of a BigTIFF with a JPEG compression automatically applied during acquisition. Images were imported into analysis software, six regions of interest were applied to various morphological locations, and the areas assessed for the color of interest. Whereas the number of designated weakly or strongly positive pixels was variable across the triplicate scans for the individual regions of interest, the total number of positive pixels was consistent. These results suggest that total positivity for a specific color representing a histochemical or immunohistochemical stain can be adequately quantitated on compressed images, but degrees of positivity (e.g., weak vs. strong) may not be as reliable. However, it is important to assess individual whole-slide imagers, file compression level and algorithm, and analysis software for reproducibility.