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The plasticity of AML drives poor clinical outcomes and confounds its longitudinal detection. However, the immediate impact of treatment on the leukemic and non-leukemic cells of the bone marrow and blood remains relatively understudied. Here, we conducted a pilot study of high dimensional longitudinal monitoring of immunophenotype in AML. To characterize changes in cell phenotype before, during, and immediately after induction treatment, we developed a 27-antibody panel for mass cytometry focused on surface diagnostic markers and applied it to 46 samples of blood or bone marrow tissue collected over time from 5 AML patients. Central goals were to determine whether changes in AML phenotype would be captured effectively by cytomic tools and to implement methods for describing the evolving phenotypes of AML cell subsets. Mass cytometry data were analyzed using established computational techniques. Within this pilot study, longitudinal immune monitoring with mass cytometry revealed fundamental changes in leukemia phenotypes that occurred over time during and after induction in the refractory disease setting. Persisting AML blasts became more phenotypically distinct from stem and progenitor cells due to expression of novel marker patterns that differed from pre-treatment AML cells and from all cell types observed in healthy bone marrow. This pilot study of single cell immune monitoring in AML represents a powerful tool for precision characterization and targeting of resistant disease.

Large B-cell lymphomas with IGH@BCL2 and MYC rearrangement, known as double-hit lymphoma (DHL), are clinically aggressive neoplasms with a poor prognosis. Some large B-cell lymphomas have concurrent abnormalities of MYC and BCL2 other than coexistent translocations. Little is known about patients with these lymphomas designated here as atypical DHL. We studied 40 patients of atypical DHL including 21 men and 19 women, with a median age of 60 years. Nine (23%) patients had a history of B-cell non-Hodgkin lymphoma. There were 30 diffuse large B-cell lymphoma (DLBCL), 7 B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma, and 3 DLBCL with coexistent follicular lymphoma. CD10, BCL2, and MYC were expressed in 28/39 (72%), 33/35 (94%), and 14/20 (70%) cases, respectively. Patients were treated with standard (n=14) or more aggressive chemotherapy regimens (n=17). We compared the atypical DHL group with 76 patients with DHLand 35 patients with DLBCL lacking MYC and BCL2 abnormalities. The clinicopathologic features and therapies were similar between patients with atypical and typical DHL. The overall survival of patients with atypical double-hit lymphoma was similar to that of patients with double-hit lymphoma (P=0.47) and significantly worse than that of patients with DLBCL with normal MYC and BCL2 (P=0.02). There were some minor differences. Cases of atypical double-hit lymphoma more often have DLBCL morphology (P<0.01), less frequently expressed CD10 (P<0.01), and patients less often had an elevated serum lactate dehydrogenase level (P=0.01). In aggregate, these results support expanding the category of MYC/BCL2 DHL to include large B-cell lymphomas with coexistent MYC and BCL2 abnormalities other than concurrent translocations.

Background The spectrum of illness and predictors of severity among children with SARS-CoV-2 infection are incompletely understood. Methods Active surveillance was performed for SARS-CoV-2 by polymerase chain reaction among symptomatic pediatric patients in a quaternary care academic hospital laboratory beginning March 12, 2020. We obtained sociodemographic and clinical data 5 (+/-3) and 30 days after diagnosis via phone follow-up and medical record review. Logistic regression was used to assess predictors of hospitalization. Results The first 1000 symptomatic pediatric patients were diagnosed in our institution between March 13, 2020 and September 28, 2020. Cough (52 %), headache (43 %), and sore throat (36 %) were the most common symptoms. Forty-one (4 %) were hospitalized; 8 required ICU admission, and 2 required mechanical ventilation (< 1 %). One patient developed multisystem inflammatory syndrome in children; one death was possibly associated with SARS-CoV-2 infection. Symptom resolution occurred by follow-up day 5 in 398/892 (45 %) patients and by day 30 in 443/471 (94 %) patients. Pre-existing medical condition (OR 7.7; 95 % CI 3.9–16.0), dyspnea (OR 6.8; 95 % CI 3.2–14.1), Black race or Hispanic ethnicity (OR 2.7; 95 % CI 1.3–5.5), and vomiting (OR 5.4; 95 % CI 1.2–20.6) were the strongest predictors of hospitalization. The model displayed excellent discriminative ability (AUC = 0.82, 95 % CI 0.76–0.88, Brier score = 0.03). Conclusions In 1000 pediatric patients with systematic follow-up, most SARS-CoV-2 infections were mild, brief, and rarely required hospitalization. Pediatric predictors of hospitalization included comorbid conditions, Black race, Hispanic ethnicity, dyspnea and vomiting and were distinct from those reported among adults.

Patients with double-hit or triple-hit lymphoma have a significantly worse prognosis compared to patients with diffuse large B-cell lymphoma without MYC rearrangement. However, the prognostic importance of extra copies of MYC , BCL2, or BCL6 has not been fully explored. We studied 663 patients with de novo diffuse large B-cell lymphoma in whom the status of MYC /8q24, BCL2 /18q21, and BCL6 /3q27 were assessed by fluorescence in situ hybridization. Cases of double or triple extra copy lymphoma were defined by the presence of increased MYC copies and increased BCL2 and/or BCL6 copies or rearrangement. In total, 76 patients with diffuse large B-cell lymphoma had MYC extra copies including 43 cases of double or triple extra copy lymphoma; 105 patients had diffuse large B-cell lymphoma with MYC -R including 56 double- or triple-hit lymphoma; and 482 diffuse large B-cell lymphoma patients had no MYC abnormality ( MYC normal). Patients with MYC extra copies, similar to MYC -R, had a worse overall survival compared with MYC normal patients (both P <0.01). The prognosis between patients with MYC extra copies and MYC -R was not statistically significantly different ( P =0.086). Cell-of-origin classification failed to correlate with survival in the MYC extra copies group, similar to the MYC -R patient group. Compared with patients with double- or triple-hit lymphoma, patients with double or triple extra copy lymphoma had a higher complete remission rate ( P =0.02), but there was no significant statistical difference in overall survival ( P =0.089). Intensive induction chemotherapy regimens improved the overall survival of patients with double or triple extra copy lymphoma, but there was no significant improvement of overall survival in patients with MYC -R tumors. Multivariate analysis showed that MYC extra copy in diffuse large B-cell lymphoma is an independent poor prognostic factor, similar to MYC rearrangement.

PCR-based diagnostics generally require nucleic acid extraction from patient specimens prior to amplification. As highlighted early in the COVID-19 pandemic, extraction steps may be difficult to scale during times of massive demand and limited reagent supply. Forgoing an extraction step, we previously reported that the N1 primer/probe-set of the widespread CDC COVID-19 assay maintains high categorical sensitivity (95%) and specificity (100%) with direct inoculation of viral transport media (VTM) into qRT-PCR reactions. In contrast, the N2 set demonstrated a prominent C t delay and low sensitivity (33%) without extraction. In the current study, we have improved the performance of this modified CDC assay (in particular the N2 set) by incorporating N1/N2/RNase P multiplexing and dissecting the effects of annealing temperature, VTM interference, and inoculum volume. The latter two factors exerted a more prominent effect on the performance of N2 than N1, although these effects were largely overcome through elevated annealing temperature. This unextracted/multiplex protocol was evaluated with 41 SARS-CoV-2 positive and 43 negative clinical samples, demonstrating a categorical sensitivity of 92.7% and specificity of 100% versus the unmodified CDC methodology. Overall, this work offers a generalizable strategy to maximize testing capabilities for COVID-19 or other emerging pathogens when resources are constrained.

Optimizing a clinical flow cytometry panel can be a subjective process dependent on experience. We develop a quantitative method to make this process more rigorous and apply it to B lymphoblastic leukemia/lymphoma (B-ALL) minimal residual disease (MRD) testing. We retrospectively analyzed our existing three-tube, seven-color B-ALL MRD panel and used our novel method to develop an optimized one-tube, eight-color panel, which was tested prospectively. The optimized one-tube, eight-color panel resulted in greater efficiency of time and resources with no loss in diagnostic power. Constructing a flow cytometry panel using a rigorous, objective, quantitative method permits optimization and avoids problems of interdependence and redundancy in a large, multiantigen panel.

Rearrangement of MYC with immunoglobulin genes is a hallmark of Burkitt lymphoma. However, this rearrangement is not entirely specific and is often accompanied by varying numbers of additional cytogenetic abnormalities. This study aimed to assess the impact of karyotypic complexity, in correlation with comprehensive immunophenotypic analyses on the diagnosis and clinical outcomes of 34 cases of MYC-IG rearranged lymphomas that included Burkitt lymphoma (twenty-two cases), diffuse large B-cell lymphoma (three cases), unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (six cases), and plasmablastic lymphoma (three cases). Additional cytogenetic abnormalities were observed in 26 of 34 cases (76%), including four cases (12%) that harbored dual translocations involving BCL-2 or BCl-6 . Burkitt lymphoma cases had a significantly lower number of additional abnormalities (mean of 1.7), compared with unclassified B-cell lymphoma (3.3), diffuse large B-cell lymphoma (21.7), and plasmablastic lymphoma (6.7). Cases with simple karyotype (⩽2 additional abnormalities) were more likely to have a diagnosis of Burkitt lymphoma (89 versus 33% in patients with >2 additional abnormalities, P <0.01) and express bcl-6 (95 versus 47%, P <0.01). In addition, Burkitt lymphoma, bcl-6 expression, and simple karyotype were individual predictors of better overall survival. However, in multivariate analyses, only bcl-6 expression remained an independent predictor, although survival could be further stratified by karyotypic complexity in bcl-6(+) patients. We conclude that simple karyotype and bcl-6 expression suggest a diagnosis of Burkitt lymphoma and may portend better overall survival. These results may be very useful in the diagnosis and stratification of MYC-IG rearranged high-grade B-cell lymphomas.

Abstract Objectives Acute myeloid leukemia (AML) is classified in part by recurrent cytogenetic abnormalities, often detected by both fluorescent in situ hybridization (FISH) and karyotype. The goal of this study was to assess the utility of FISH and karyotyping at diagnosis and follow-up. Methods Adult AML samples at diagnosis or follow-up with karyotype and FISH were identified. Concordance was determined, and clinical characteristics and outcomes for discordant results were evaluated. Results Karyotype and FISH results were concordant in 193 (95.0%) of 203 diagnostic samples. In 10 cases, FISH detected an abnormality, but karyotype was normal. Of these, one had a FISH result with clinical significance. In follow-up cases, 17 (8.1%) of 211 showed FISH-positive discordant results; most were consistent with low-level residual disease. Conclusions Clinically significant discordance between karyotype and AML FISH is uncommon. Consequently, FISH testing can safely be omitted from most of these samples. Focused FISH testing is more useful at follow-up, for minimal residual disease detection.

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. 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. 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). 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. 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.

Training individuals to make accurate decisions from medical images is a critical component of education in diagnostic pathology. We describe a joint experimental and computational modeling approach to examine the similarities and differences in the cognitive processes of novice participants and experienced participants (pathology residents and pathology faculty) in cancer cell image identification. For this study we collected a bank of hundreds of digital images that were identified by cell type and classified by difficulty by a panel of expert hematopathologists. The key manipulations in our study included examining the speed-accuracy tradeoff as well as the impact of prior expectations on decisions. In addition, our study examined individual differences in decision-making by comparing task performance to domain general visual ability (as measured using the Novel Object Memory Test (NOMT) (Richler et al. Cognition 166:42–55, 2017). Using signal detection theory and the diffusion decision model (DDM), we found many similarities between experts and novices in our task. While experts tended to have better discriminability, the two groups responded similarly to time pressure (i.e., reduced caution under speed instructions in the DDM) and to the introduction of a probabilistic cue (i.e., increased response bias in the DDM). These results have important implications for training in this area as well as using novice participants in research on medical image perception and decision-making.