Mimicry of inherited red cell disorders: the result of somatic mutations in a clonal myeloid disease.

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Seniors from United States allopathic medical schools matching into pathology residency, 2018-2022.

BACKGROUND: From 2008 to 2017, 28.8 % fewer United States allopathic medical students (MD seniors) applied to pathology residency in the Main Residency Match (MRM) and 27.5 % fewer matched. This study is a 5-year follow-up. METHODS: MRM data from 2018 to 2022 were reviewed to determine the numbers of MD seniors that applied and matched to pathology residency and other major medical specialties. RESULTS: From 2018 to 2022, the number of MD seniors applying to pathology increased 4.6 % from 237 to 248, while MD seniors matching to pathology increased 5.0 % from 220 to 231. For the 4 years from 2018 to 2021, there was a slight decline in MD seniors filling pathology positions, followed by a substantial 16.7 % spike in 2022. For the entire 5-year interval, because the number of filled pathology residency positions increased by 9.0 %, the percentage of filled positions taken by MD seniors declined from 38.7 % to 37.3 %. Of the 15 major medical specialties evaluated, pathology now has the 14th lowest percentage of filled positions taken by MD seniors. CONCLUSIONS: The number of MD seniors applying and matching to pathology residency increased over the past 5-years, in contrast to the timespan of 2008 to 2017. However, the percentage of pathology residency positions taken by MD seniors continued to decline and remains low compared to other major medical specialties. MRM data should be continually monitored to study trends in MD seniors filling pathology residency positions in the context of new recruitment efforts and the pandemic.

Clonal cytopenia of undetermined significance (CCUS) with dysplasia is enriched for MDS-type molecular findings compared to CCUS without dysplasia.

OBJECTIVES: Although morphologic dysplasia is not typically considered a feature of CCUS, we have consistently observed low-level bone marrow (BM) dysplasia among CCUS patients. We sought to determine whether sub-diagnostic BM dysplasia in CCUS patients is associated with other clinico-pathologic findings of myelodysplastic syndrome (MDS). METHODS: We identified 49 CCUS patients, 25 with sub-diagnostic dysplasia (CCUS-D), and 24 having no dysplasia (CCUS-ND). We compared the clinical, histologic, and laboratory findings of CCUS-D and CCUS-ND patients to 49 MDS patients, including blood cell counts, BM morphology, flow cytometry, cytogenetics, and results of next-generation sequencing. RESULTS: No statistically significant differences were observed between CCUS-D and CCUS-ND patients in the degree of cytopenias, BM cellularity, myeloid-to-erythroid ratio, or the presence of flow cytometric abnormalities. However, compared to CCUS-ND, CCUS-D patients exhibited increased mutations in myeloid malignancy-associated genes, including non-TET2/DNMT3A/ASXL1 variants, spliceosome (SF3B1, SRSF2, ZRSR2, or U2AF1) variants, and IDH2/RUNX1/CBL variants. CCUS-D patients were also enriched for higher variant allele frequencies and co-mutation of TET2/DNMT3A/ASXL1 with other genes. CONCLUSIONS: CCUS-D patients exhibit a molecular (but not clinical) profile more similar to MDS patients than CCUS-ND, suggesting CCUS-D may represent a more immediate precursor to MDS and may warrant closer clinical follow-up.

The medical student's guide to pathology residency, fellowships, and careers.

Recent studies have shown that relatively few MD, DO, and underrepresented in medicine (URM) students and physicians are matching into pathology residency in the United States (US). In the 2021 Main Residency Match, just 33.6% of filled pathology residency positions were taken by senior year students at US allopathic medical schools. This has been attributed to the fact that pathology is not a required rotation in most US medical schools, pathology is often taught in an integrated curriculum in the US where is does not stand out as a distinct field, and because the COVID-19 pandemic led to a suspension of in-person pathology rotations and electives. Ultimately, many US medical students fail to consider pathology as a career pathway. The objective of this article is to provide medical students with basic information, in the form of frequently asked questions (FAQs), about pathology training and career opportunities. This was accomplished by forming a team of MD and DO pathology attendings, pathology trainees, and a medical student from multiple institutions to create a pathology guide for medical students. This guide includes information about post-sophomore fellowships, 5 major pathology residency tracks, more than 20 fellowship pathways, and allopathic and osteopathic board examinations. This guide also contains photographs and descriptions of major pathology sub-specialties, including the daily and on-call duties and responsibilities of pathology residents. The exciting future of pathology is also discussed. This guide supports the agenda of the College of American Pathologists' (CAP) Pathologist Pipeline Initiative to improve student recruitment into pathology.

"Dual-gene" malaria-resistance: Therapeutically-rational exchange (T-REX) of group-O sickle trait and group-O C-traittrait red blood cells can be evaluated in Benin and Nigeria.

BACKGROUND: Using indicators of disease severity, clinicians can predict which Plasmodium falciparum (Pf) malaria patients being treated with artesunate or quinine are likely to die despite these drugs. Effective "rescue adjuncts" are needed when drugs alone are inadequate. "Therapeutically-rational exchange" (T-REX) of special malaria-resistant red blood cells (RBCs) has been proposed to optimize adjunctive exchange transfusion. METHODS: Studies were reviewed that (1) quantified how group-O status and "sickle-trait" (HbAS) and "C-trait" (HbAC) hemoglobins affect Pf mortality, risk of thrombosis, or birth outcomes for women with pregnancy associated malaria (PAM), (2) reported prevalences of "dual-gene" malaria-resistant RBCs, or (3) reflected the level of exchange-transfusion and malaria-related expertise in Benin and Nigeria. RESULTS: Data show that the malaria- and thrombosis-resistance of RBCs depend on specific genes and the patient's clinical status and medical history. In malaria-endemic Benin and Nigeria, prevalences of "dual-gene" malaria-resistant group-O HbAS and group-O HbAC RBCs are substantial, and both malaria- and exchange-related expertise are outstanding. CONCLUSIONS: T-REX of "dual-gene" malaria-resistant RBCs is feasible in Benin and Nigeria and warrants evaluation as a rescue adjunct for 3 subsets of Pf-malaria patients. For therapeutic use, group-O HbAS RBCs are likely to be more effective than non-O HbAS RBCs for Pf-infected patients who (1) have a history of thrombosis or (2) are taking birth-control hormones while group-O HbAC RBCs may substantially improve birth outcomes for women with PAM. Studies suggest it is prudent to assume - until proven otherwise - that T-REX of "dual-gene" malaria-resistant RBCs can improve ("personalize") rescue of these patient subsets.

Pathology's historic 2019 incoming residents: Why "the internationalization of pathology" may markedly advance transfusion medicine and cellular therapeutics.

OBJECTIVES: This study had two objectives: (1) to determine if, in the United States of America (US), the proportion of non-US citizen international medical graduates (non-US IMGs) entering pathology residencies had increased (again) in 2019 and (2) to assess how this multi-year trend might impact transfusion medicine in the US. METHODS: The most recent (2019) "National Resident Matching Program" (NRMP) data were analyzed. To assess potential future impact, using controversies related to Plasmodium falciparum (Pf) malaria, conflicting US and non-US perspectives were reviewed. Differences between published US and non-US views were identified regarding, for example, the value of Pf-resistant ("variant") red blood cells (RBCs) and exchange transfusions. RESULTS: Year 2019 is the first year non-US IMGs were the largest group to fill residency-training positions for a major US specialty via the "Main Residency Match." Also notable, US and non-US views were found to differ markedly regarding (1) the value and safety of Pf-resistant RBC variants and exchange transfusions, and (2) the threat of drug-resistant Pf-malaria parasites. Non-US clinicians and researchers seem more concerned about Pf-malaria, and their interest in cellular therapies seems greater and more optimistic. CONCLUSIONS: In 2019, the historically high proportion of non-US IMGs among incoming pathology residents dramatically highlights the steady demographic shift that began years ago: "the internationalization of pathology" in the US. Fortunately, a review of publications related to exchange transfusion, Pf-malaria, and variant RBCs suggests non-US IMGs may markedly promote and advance cell therapies such as therapeutically-rational exchange (T-REX) of disease-resistant RBCs.

Resolving Discrepancies in Idylla BRAF Mutational Assay Results Using Targeted Next-Generation Sequencing.

BRAF mutation identification is important for the diagnosis and treatment of several tumor types, both solid and hematologic. Rapid identification of BRAF mutations is required to determine eligibility for targeted BRAF inhibitor therapy. The Idylla BRAF mutation assay is a rapid, multiplex allele-specific PCR test designed to detect the most common oncogenic BRAF V600 mutations in formalin-fixed paraffin-embedded (FFPE) tissue samples. Here, we describe the validation of the Idylla BRAF mutation assay in our laboratory. During routine clinical practice, we noticed cases in which BRAF V600 mutations were identified with unusual amplification curves, with three cases displaying a delayed amplification within a double amplification pattern and two false-positive calls. We therefore initiated a quality improvement effort to systematically and retrospectively evaluate next-generation sequencing (NGS)-tested cases with BRAF mutations identified within five amino acids of BRAF codon V600 and did not identify additional false-positive cases. We hypothesize that late amplification in a double amplification pattern may represent non-specific amplification, whereas cases displaying single delayed amplification curves may stem from the presence of either non-V600 variants, very low-level V600 variants, cytosine deamination artifacts, and/or non-specific amplification by an allele-specific PCR primer. Regardless, we recommend that Idylla BRAF cases with non-classical amplification curves undergo reflex NGS testing. These findings are likely relevant for other Idylla assays interrogating hotspot mutations in genes such as EGFR, IDH1/2, KRAS, and NRAS.

Clonally unrelated primary large B-cell lymphomas separated by over a decade involving the central nervous system and testicle: Possible predisposition to lymphomas of immune-privileged sites?

Primary large B-cell lymphomas of immune-privileged sites (IP-LBCLs) comprise LBCLs arising within "immune sanctuaries," including the central nervous system (CNS), vitreoretina, and testes. Although patients present with localized disease, the prognosis remains poor with high relapse rates, either at the originating site or within another immune-privileged site. Generally, in the presence of an antecedent IP-LBCL, subsequent LBCLs are expected to be clonally related. However, we present a primary CNS LBCL and later primary testicular LBCL in a middle-aged man, diagnosed over a decade apart, which proved to be clonally unrelated by targeted ultra-deep next-generation sequencing of the IgH locus.

ABO blood group antigens and differential glycan expression: Perspective on the evolution of common human enzyme deficiencies.

Enzymes catalyze biochemical reactions and play critical roles in human health and disease. Enzyme variants and deficiencies can lead to variable expression of glycans, which can affect physiology, influence predilection for disease, and/or directly contribute to disease pathogenesis. Although certain well-characterized enzyme deficiencies result in overt disease, some of the most common enzyme deficiencies in humans form the basis of blood groups. These carbohydrate blood groups impact fundamental areas of clinical medicine, including the risk of infection and severity of infectious disease, bleeding risk, transfusion medicine, and tissue/organ transplantation. In this review, we examine the enzymes responsible for carbohydrate-based blood group antigen biosynthesis and their expression within the human population. We also consider the evolutionary selective pressures, e.g. malaria, that may account for the variation in carbohydrate structures and the implications of this biology for human disease.

Clonal cytopenia of undetermined significance (CCUS)-associated reversion of donor-derived, transient αß T-cell large granular clonal lymphocytosis, emerging post-transplant in a patient with a history of γδ T-cell large granular lymphocytic leukemia.

Autologous and allogeneic hematopoietic stem cell transplantation (HSCT) has revolutionized the therapy of hematolymphoid malignancies. Yet, how to best detect or predict the emergence of HSCT-related complications remain unresolved. Here, we describe a case of donor-derived, transient Alpha Beta (αß) T-cell large granular clonal lymphocytosis and cytopenia that emerged post-HSCT in a patient with a history of gamma delta (γδ) T-cell large granular lymphocytic leukemia (T-LGLL). Clonal unrelatedness of post-transplant T-LGL lymphocytosis to the patient's pretransplant T-LGLL was first identified by T-cell receptor (TCR) PCR showing different sized fragments of rearranged gamma chains, in addition to shift from γδ to αß TCR expression by flow cytometry analyses. Donor-derivation of the patient's post-transplant clonal lymphocytosis was confirmed by serial chimerism analyses of recipient's blood specimens demonstrating 100% donor DNA. Moreover, oncogenic DNMT3A and RUNX1 mutations were detected by next-generation sequencing (NGS) only in post-transplant specimens. Intriguingly, despite continued increase in DNMT3A and RUNX1 mutation load, the patient's clonal lymphocytosis and anemia eventually largely resolved; yet, the observed mutation profile with persistent thrombocytopenia indicated secondary clonal cytopenia of undetermined significance (CCUS) in the absence of overt morphologic evidence of myeloid neoplasm in the marrow. This case illustrates the utility of longitudinal chimerism analysis and NGS testing combined with flow cytometric immunophenotyping to evaluate emerging donor-derived hematolymphoid processes and to properly interpret partial functional engraftment. It may also support the notion that driver mutation-induced microenvironmental changes may paradoxically contribute to reestablishing tissue homeostasis.

Current clinical practices and challenges in molecular testing: a GOAL Consortium Hematopathology Working Group report.

While molecular testing of hematologic malignancies is now standard of care, there is variability in practice and testing capabilities between different academic laboratories, with common questions arising on how to best meet clinical expectations. A survey was sent to hematopathology subgroup members of the Genomics Organization for Academic Laboratories consortium to assess current and future practice and potentially establish a reference for peer institutions. Responses were received from 18 academic tertiary-care laboratories regarding next-generation sequencing (NGS) panel design, sequencing protocols and metrics, assay characteristics, laboratory operations, case reimbursement, and development plans. Differences in NGS panel size, use, and gene content were reported. Gene content for myeloid processes was reported to be generally excellent, while genes for lymphoid processes were less well covered. The turnaround time (TAT) for acute cases, including acute myeloid leukemia, was reported to range from 2 to 7 calendar days to 15 to 21 calendar days, with different approaches to achieving rapid TAT described. To help guide NGS panel design and standardize gene content, consensus gene lists based on current and future NGS panels in development were generated. Most survey respondents expected molecular testing at academic laboratories to continue to be viable in the future, with rapid TAT for acute cases likely to remain an important factor. Molecular testing reimbursement was reported to be a major concern. The results of this survey and subsequent discussions improve the shared understanding of differences in testing practices for hematologic malignancies between institutions and will help provide a more consistent level of patient care.

Incidental discovery of acute myeloid leukemia during liquid biopsy of a lung cancer patient.

Liquid biopsy is considered an alternative to standard next-generation sequencing (NGS) of solid tumor samples when biopsy tissue is inadequate for testing or when testing of a peripheral blood sample is preferred. A common assumption of liquid biopsies is that the NGS data obtained on circulating cell-free DNA is a high-fidelity reflection of what would be found by solid tumor testing. Here, we describe a case that challenges this widely held assumption. A patient diagnosed with lung carcinoma showed pathogenic IDH1 and TP53 mutations by liquid biopsy NGS at an outside laboratory. Subsequent in-house NGS of a metastatic lymph node fine-needle aspiration (FNA) sample revealed two pathogenic EGFR mutations. Morphologic and immunophenotypic assessment of the patient's blood sample identified acute myeloid leukemia, with in-house NGS confirming and identifying pathogenic IDH1, TP53, and BCOR mutations, respectively. This case, together with a few similar reports, demonstrates that caution is needed when interpreting liquid biopsy NGS results, especially if they are inconsistent with the presumptive diagnosis. Our case suggests that routine parallel sequencing of peripheral white blood cells would substantially increase the fidelity of the obtained liquid biopsy results.

Identification of a Cancer-Predisposing Germline POT1 p.Ile49Metfs*7 Variant by Targeted Sequencing of a Splenic Marginal Zone Lymphoma.

Germline disruptive variants in Protection of Telomeres 1 (POT1) predispose to a wide variety of cancers, including melanoma, chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, myeloproliferative neoplasms, and glioma. We report the first case of splenic marginal zone lymphoma (SMZL) arising in a patient with a germline POT1 variant: a 65-year-old male with an extensive history of cancer, including melanoma and papillary thyroid carcinoma, who presented with circulating atypical lymphocytosis. Bone marrow biopsy revealed 20% involvement by a CD5-CD10- B-cell lymphoma that was difficult to classify. During the clinical workup of his low-grade lymphoma, targeted next-generation sequencing (NGS) identified POT1 p.I49Mfs*7 (NM_015450:c. 147delT) at a variant allele frequency (VAF) of 51%. NGS of skin fibroblasts confirmed the POT1 variant was germline. This likely pathogenic POT1 loss-of-function variant has only been reported once before as a germline variant in a patient with glioma and likely represents one of the most deleterious germline POT1 variants ever linked to familial cancer. The spectrum of cancers associated with germline pathogenic POT1 variants (i.e., autosomal dominant POT1 tumor predisposition syndrome) should potentially be expanded to include SMZL, a disease often associated with the loss of chromosome 7q: the location of the POT1 genetic locus (7q31.33).