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PURPOSE: This study investigated the effectiveness of algorithmic testing in hematopathology at the Brigham and Women's Hospital and Dana-Farber Cancer Institute (DFCI). The algorithm was predicated on test selection after an initial pathologic evaluation to maximize cost-effective testing, especially for expensive molecular and cytogenetic assays. MATERIALS AND METHODS: Standard ordering protocols (SOPs) for 17 disease categories were developed and encoded in a decision support application. Six months of retrospective data from application beta testing was obtained and compared with actual testing practices during that timeframe. In addition, 2 years of prospective data were also obtained from patients at one community satellite site. RESULTS: A total of 460 retrospective cases (before introduction of algorithmic testing) and 109 prospective cases (following introduction) were analyzed. In the retrospective data, 61.7% of tests (509 of 825) were concordant with the SOPs while 38.3% (316 of 825) were overordered and 30.8% (227 of 736) of SOP-recommended tests were omitted. In the prospective data, 98.8% of testing was concordant (244 of 247 total tests) with only 1.2% overordered tests (3 of 247) and 7.6% omitted tests (20 of 264 SOP-recommended tests; overall P < .001). The cost of overordered tests before implementing SOP indicates a potential annualized saving of $1,347,520 in US dollars (USD) in overordered testing at Brigham and Women's Hospital/DFCI. Only two of 316 overordered tests (0.6%) returned any additional information, both for extremely rare clinical circumstances. CONCLUSION: Implementation of SOPs dramatically improved test ordering practices, with a just right number of ancillary tests that minimizes cost and has no significant impact on acquiring key informative test results.
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Médula Ósea , Hospitales , Humanos , Femenino , Médula Ósea/patología , Estudios Retrospectivos , Biología MolecularRESUMEN
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.
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To better characterize the heterogeneity of multiple myeloma (MM), we profiled plasma cells (PCs) and their B cell lymphopoiesis in the BM samples from patients with monoclonal gammopathy of undetermined significance, smoldering MM, and active MM by mass cytometry (CyTOF) analysis. Characterization of intra- and interneoplastic heterogeneity of malignant plasmablasts and PCs revealed overexpression of the MM SET domain (MMSET), Notch-1, and CD47. Variations in upregulation of B cell signaling regulators (IFN regulatory factor 4 [IRF-4], CXCR4, B cell lymphoma 6 [Bcl-6], c-Myc, myeloid differentiation primary response protein 88 [MYD88], and spliced X box-binding protein 1 [sXBP-1]) and aberrant markers (CD319, CD269, CD200, CD117, CD56, and CD28) were associated with different clinical outcomes in clonal PC subsets. In addition, prognosis was related to heterogeneity in subclonal expression of stemness markers, including neuroepithelial stem cell protein (Nestin), SRY-box transcription factor 2 (Sox2), Krüppel-like factor 4 (KLF-4), and Nanog. Furthermore, we have defined significantly elevated levels of MMSET, MYD88, c-Myc, CD243, Notch-1, and CD47 from hematopoietic stem cells to PCs in myeloma B cell lymphopoiesis, noted even in premalignant conditions, with variably modulated expression of B cell development regulators, including IRF-4, Bcl-2, Bcl-6, and sXBP-1; aberrant PC markers (such as CD52, CD44, CD200, CD81, CD269, CD117, and CXCR4); and stemness-controlling regulators, including Nanog, KLF-4, octamer-binding transcription factor 3/4 (Oct3/4), Sox2, and retinoic acid receptor α2 (RARα2). This study provides the rationale for precise molecular profiling of patients with MM by CyTOF technology to define disease heterogeneity and prognosis.
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Mieloma Múltiple , Humanos , Mieloma Múltiple/patología , Antígeno CD47/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Linfopoyesis , Linfocitos B/metabolismoRESUMEN
Novel drug discoveries have shifted the treatment paradigms of most hematological malignancies, including multiple myeloma (MM). However, this plasma cell malignancy remains incurable, and novel therapies are therefore urgently needed. Whole-genome transcriptome analyses in a large cohort of MM patients demonstrated that alterations in pre-mRNA splicing (AS) are frequent in MM. This manuscript describes approaches to identify disease-specific alterations in MM and proposes RNA-based therapeutic strategies to eradicate such alterations. As a "proof of concept", we examined the causes of aberrant HMMR (Hyaluronan-mediated motility receptor) splicing in MM. We identified clusters of single nucleotide variations (SNVs) in the HMMR transcript where the altered splicing took place. Using bioinformatics tools, we predicted SNVs and splicing factors that potentially contribute to aberrant HMMR splicing. Based on bioinformatic analyses and validation studies, we provided the rationale for RNA-based therapeutic strategies to selectively inhibit altered HMMR splicing in MM. Since splicing is a hallmark of many cancers, strategies described herein for target identification and the design of RNA-based therapeutics that inhibit gene splicing can be applied not only to other genes in MM but also more broadly to other hematological malignancies and solid tumors as well.
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Neoplasias Hematológicas , Mieloma Múltiple , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Empalme Alternativo , ARN , Empalme del ARNRESUMEN
Waldenström macroglobulinemia (WM) is a rare subtype of non-Hodgkin lymphoma characterized by malignant lymphoplasmacytic cells in the bone marrow (BM). To dissect the pathophysiology of WM, we evaluated clonal cells by mapping of B cell lymphomagenesis with adaptive and innate immune tumor microenvironment (TME) in the BM of WM patients using mass cytometry (CyTOF). In-depth immunophenotypic profiling of WM cells exhibited profound expansion of clonal cells in both unswitched and switched memory B cells and also plasma cells with aberrant expression variations. WM B lymphomagenesis was associated with reduction of most B cell precursors assessed with the same clonally restricted light chain and phenotypic changes. The immune TME was infiltrated by mature monocytes, neutrophils and adaptive T cells, preferentially subsets of effector T helper, effector CTL and effector memory CTL cells that were associated with superior overall survival (OS), in contrast to progenitors of T cells and myeloid/monocytic lineage subsets that were suppressed in WM cohort. Moreover, decrease in immature B and NKT cells was related to worse OS in WM patients. Innate and adaptive immune subsets of WM TME were modulated by immune checkpoints, including PD-1/PD-L1&PD-L2, TIGIT/PVR, CD137/CD137-L, CTLA-4, BTLA and KIR expression. The response of ibrutinib treatment to the reduction of clonal memory B cell was associated with high levels of immature B cells and effector memory CTL cells. Our study demonstrates that CyTOF technology is a powerful approach for characterizing the pathophysiology of WM at various stages, predicting patient risk and monitoring the effectiveness of treatment strategies.
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Linfoma de Células B , Macroglobulinemia de Waldenström , Humanos , Macroglobulinemia de Waldenström/tratamiento farmacológico , Macroglobulinemia de Waldenström/metabolismo , Microambiente Tumoral , Células Plasmáticas/patología , Linfocitos B/patologíaRESUMEN
Aggressive subtypes of non-Hodgkin lymphoma may uncommonly be referred to clinical oncologists for treatment of acute leukemia, due to an elevated or rapidly rising white blood cell count (WBC), with circulating neoplastic cells that morphologically resemble leukemic blasts seen in acute myeloid or lymphoblastic leukemia. We describe six cases of non-Hodgkin lymphoma that mimicked acute leukemia and were identified in the pathology records of the Brigham and Women's Hospital. The patients were older adults (mean age 70 years), who presented with leukocytosis (mean 79.7 × 109/L) with circulating neoplastic cells (mean 57%), which mimicked leukemic blasts, thrombocytopenia, and anemia (4/6 patients). In each case, immunophenotypic analysis identified a population of mature B cells or mature T cells. We identified 15 additional cases of non-Hodgkin lymphoma in the literature that mimicked acute leukemia; considering all 21 cases, 11 had an appearance of acute lymphoblastic leukemia, 4 had an appearance of acute monocytic leukemia, and 6 had an appearance of acute leukemia unable to be further categorized. In general, patients exhibited poor overall survival. These cases illustrate the importance of comprehensive immunophenotypic analysis in the initial evaluation of hematolymphoid neoplasms, and that occasional cases of non-Hodgkin lymphomas can resemble acute leukemia at initial presentation.
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Oncogenic activated RAS mutations have been detected in 50% of de novo and 70% of relapsed multiple myeloma (MM) patients. Translocation t(11;14) involving IgH/CCDN1 and overexpression of cyclin-Ds are early events in MM pathogenesis, enhancing uncontrolled MM cell growth. We hypothesized that targeting both RAS/MAPK pathway molecules including Erk1/2 along with cyclin-Ds enhances MM cytotoxicity and minimizes side effects. Recent studies have demonstrated the high potency of Erk1/2 and CDK4/6 inhibitors in metastatic relapsed cancers, and here we tested anti-MM effects of the Erk1/2 + CDK4/6 inhibitor combination. Our studies showed strong synergistic (IC < 0.5) cytotoxicity of Erk1/2i + CDK4/6i in MM-cells. Erk1/2i + CDK4/6i treatment in a dose-dependent manner arrested MM-cells in the G0/G1 phase and activated mitochondrial apoptotic signaling. Our studies showed that Erk1/2i + CDK4/6i treatment-induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling, such as c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of these inhibitors. We identified Erk1/2i + CDK4/6i treatment associated five-gene signature which includes SNRPB and SLC25A5; these genes are involved in RNA processing and mitochondrial metabolism, respectively. Overall, our studies provide the preclinical framework for Erk1/2i + CDK4/6i combination clinical trials to target Ras+CDK pathways to improve patient outcome in MM.
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Neoplasias de la Mama , Mieloma Múltiple , Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Femenino , Humanos , Mieloma Múltiple/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
The COVID-19 pandemic has highlighted racial health disparities within the United States. Although social determinants of health are the most likely drivers of this disparity, it is possible that genetic traits enriched in the black population like sickle cell trait (SCT) could worsen the morbidity and mortality of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Patients admitted for SARS-CoV-2 infection who identified as black or African American were included in the study (n = 166). Blood remnants were tested for SCT, and clinical data were abstracted from the chart. There was no difference in mortality between those with SCT and those without. There was no difference in respiratory complications between groups, but those without SCT had a much higher burden of chronic lung disease (P = .004). Those with SCT had higher creatinine on admission (P = .004), but no difference in in-hospital renal complications (P = .532). Notably, 12% of the cohort had SCT, which is higher than the expected 7.31% (P = .025). Our study did not show any evidence of increased end organ damage, morbidity, or mortality from SARS-CoV-2 infection among patients with SCT but did show differences in admission creatinine and preexisting lung disease.
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COVID-19 , Rasgo Drepanocítico , Humanos , Morbilidad , Pandemias , SARS-CoV-2 , Estados UnidosRESUMEN
Chromosome 13q deletion [del(13q)], harboring the miR-15a/16-1 cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without del(13q), suggesting important tumor-suppressor activity. However, the role of miR-15a/16-1 in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1 cluster in murine GC B cells induces moderate but widespread molecular and functional changes including an increased number of GC B cells, percentage of dark zone B cells, and maturation into plasma cells. With time, this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM that has progressed to extramedullary disease. We corroborate human primary EP having low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that, although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1 cluster in the regulation of the GC reaction and its fundamental context-dependent tumor-suppression function in plasma cell and B-cell malignancies.
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Linfoma de Células B Grandes Difuso/genética , MicroARNs/genética , Neoplasias de Células Plasmáticas/genética , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Deleción Cromosómica , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/patología , Cromosomas Humanos Par 13/genética , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma de Células B Grandes Difuso/patología , Ratones Endogámicos C57BL , Familia de Multigenes , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Neoplasias de Células Plasmáticas/patología , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Plasmacitoma/genética , Plasmacitoma/patologíaRESUMEN
CONTEXT.: Minimal residual disease (MRD) testing by flow cytometry is ubiquitous in hematolymphoid neoplasm monitoring, especially B-lymphoblastic leukemia (B-ALL), for which it provides predictive information and guides management. Major heterogeneity was identified in 2014. Subsequently, new Flow Cytometry Checklist items required documentation of the sensitivity determination method and required lower level of detection (LLOD) inclusion in final reports. This study assesses Laboratory Accreditation Program (LAP) participation and new checklist items' impact on flow cytometry MRD testing. OBJECTIVES.: To survey flow cytometry laboratories about MRD testing for B-ALL and plasma cell myeloma. In particular, enumerate the laboratories performing MRD testing, the proportion performing assays with very low LLODs, and implementation of new checklist items. DESIGN.: Supplemental questions were distributed in the 2017-A mailing to 548 flow cytometry laboratories subscribed to the College of American Pathologists FL3 Proficiency Testing Survey (Flow Cytometry-Immunophenotypic Characterization of Leukemia/Lymphoma). RESULTS.: The percentage of laboratories performing MRD studies has significantly decreased since 2014. Wide ranges of LLOD and collection event numbers were reported for B-ALL and plasma cell myeloma. Most laboratories determine LLOD by using dilutional studies and include it in final reports; a higher proportion of LAP participants used these practices than nonparticipants. CONCLUSIONS.: Several MRD testing aspects vary among laboratories receiving FL3 Proficiency Testing materials. After the survey in 2014, new checklist items were implemented. As compared to 2014, fewer laboratories are performing MRD studies. While LLOD remains heterogeneous, a high proportion of LAP subscribers follow the new checklist requirements and, overall, target LLOD recommendations from disease-specific working groups are met.
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Ensayos de Aptitud de Laboratorios/normas , Mieloma Múltiple/diagnóstico , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Acreditación , American Medical Association , Citometría de Flujo , Estudios de Seguimiento , Humanos , Inmunofenotipificación , Mieloma Múltiple/patología , Neoplasia Residual/patología , Patólogos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Encuestas y Cuestionarios , Estados UnidosRESUMEN
CONTEXT.: The diagnostic workup of lymphoma continues to evolve rapidly as experience and discovery led to the addition of new clinicopathologic entities and techniques to differentiate them. The optimal clinically effective, efficient, and cost-effective approach to diagnosis that is safe for patients can be elusive, in both community-based and academic practice. Studies suggest that there is variation in practice in both settings. OBJECTIVE.: To develop an evidence-based guideline for the preanalytic phase of testing, focusing on specimen requirements for the diagnostic evaluation of lymphoma. DESIGN.: The American Society for Clinical Pathology, the College of American Pathologists, and the American Society of Hematology convened a panel of experts in the laboratory workup of lymphoma to develop evidence-based recommendations. The panel conducted a systematic review of literature to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, recommendations were derived based on the available evidence, strength of that evidence, and key judgements as defined in the Grading of Recommendations Assessment, Development, and Evaluation Evidence to Decision framework. RESULTS.: Thirteen guideline statements were established to optimize specimen selection, ancillary diagnostic testing, and appropriate follow-up for safe and accurate diagnosis of indolent and aggressive lymphoma. CONCLUSIONS.: Primary diagnosis and classification of lymphoma can be achieved with a variety of specimens. Application of the recommendations can guide decisions on specimen suitability, diagnostic capabilities, and correct use of ancillary testing. Disease prevalence in patient populations, availability of ancillary testing, and diagnostic goals should be incorporated into algorithms tailored to each practice environment.
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Medicina Basada en la Evidencia , Linfoma , Patólogos , Patología Clínica , Adulto , Humanos , American Medical Association , Educación , Hematología/educación , Laboratorios , Linfoma/clasificación , Linfoma/diagnóstico , Linfoma/patología , Patólogos/educación , Patología Clínica/educación , Estados Unidos , Revisiones Sistemáticas como AsuntoRESUMEN
OBJECTIVES: The diagnostic workup of lymphoma continues to evolve rapidly as experience and discovery lead to the addition of new clinicopathologic entities and techniques to differentiate them. The optimal clinically effective, efficient, and cost-effective approach to diagnosis that is safe for patients can be elusive, in both community-based and academic practice. Studies suggest that there is variation in practice in both settings. THE AIM OF THIS REVIEW IS TO: develop an evidence-based guideline for the preanalytic phase of testing, focusing on specimen requirements for the diagnostic evaluation of lymphoma. METHODS: The American Society for Clinical Pathology, the College of American Pathologists, and the American Society of Hematology convened a panel of experts in the laboratory workup of lymphoma to develop evidence-based recommendations. The panel conducted a systematic review of the literature to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach, recommendations were derived based on the available evidence, the strength of that evidence, and key judgments as defined in the GRADE Evidence to Decision framework. RESULTS: Thirteen guideline statements were established to optimize specimen selection, ancillary diagnostic testing, and appropriate follow-up for safe and accurate diagnosis of indolent and aggressive lymphoma. CONCLUSIONS: Primary diagnosis and classification of lymphoma can be achieved with a variety of specimens. Application of the recommendations can guide decisions about specimen suitability, diagnostic capabilities, and correct utilization of ancillary testing. Disease prevalence in patient populations, availability of ancillary testing, and diagnostic goals should be incorporated into algorithms tailored to each practice environment.
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Linfoma , Patología Clínica , Humanos , Análisis Costo-Beneficio , Práctica Clínica Basada en la Evidencia , Linfoma/diagnóstico , Linfoma/patología , Patología Clínica/normas , Manejo de Especímenes , Estados Unidos , Revisiones Sistemáticas como AsuntoRESUMEN
Clinical uncertainty exists regarding which assay should be designated as the standard monitoring coagulation test for intravenous unfractionated heparin (UFH). Several studies have compared the use of activated partial thromboplastin time (aPTT) and antifactor-Xa (anti-Xa) and have come out with varying results. The correlation between these 2 tests varied, markedly from strong to weak. Some have demonstrated that monitoring with anti-Xa heparin assay leads to fewer dose adjustments, resulting in fewer laboratory tests, while others have not. In the current study, we evaluated the correlation between aPTT and anti-Xa values to guide clinical management of UFH, with the intention to develop a new correlation nomogram.
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Pruebas de Coagulación Sanguínea/métodos , Heparina/uso terapéutico , Pruebas de Coagulación Sanguínea/normas , Inhibidores del Factor Xa/análisis , Femenino , Heparina/administración & dosificación , Humanos , Bombas de Infusión , Masculino , Persona de Mediana Edad , Tiempo de Tromboplastina ParcialRESUMEN
Inclusion body myositis is a late onset treatment-refractory autoimmune disease of skeletal muscle associated with a blood autoantibody (anti-cN1A), an HLA autoimmune haplotype, and muscle pathology characterized by cytotoxic CD8+ T cell destruction of myofibres. Here, we report on translational studies of inclusion body myositis patient muscle compared with a diverse set of other muscle disease samples. Using available microarray data on 411 muscle samples from patients with inclusion body myositis (n = 40), other muscle diseases (n = 265), and without neuromuscular disease (normal, n = 106), we identified a signature of T-cell cytotoxicity in inclusion body myositis muscle coupled with a signature of highly differentiated CD8 T-cell effector memory and terminally differentiated effector cells. Further, we examined killer cell lectin-like receptor G1 (KLRG1) as a marker of this population of cells, demonstrated the correlation of KLRG1 gene expression with lymphocyte cytotoxicity across 28 870 human tissue samples, and identified the presence of KLRG1 on pathogenic inclusion body myositis muscle invading T cells and an increase in KLRG1 expressing T cells in inclusion body myositis blood. We examined inclusion body myositis muscle T-cell proliferation by Ki67 immunohistochemistry demonstrating that diseased muscle-invading T cells are minimally or non-proliferative, in accordance with known properties of highly differentiated or terminally differentiated T cells. We found low expression of KLRG1 on infection-protective human lymphoid tissue central memory T cells and autoimmune-protective human blood regulatory T cells. Targeting highly differentiated cytotoxic T cells could be a favourable approach to treatment of inclusion body myositis.