Analytical Validation of an Automated Semiconductor-Based Next-Generation Sequencing Assay for Detection of DNA and RNA Alterations in Myeloid Neoplasms.

Myeloid neoplasms are heterogeneous tumors derived from early hematopoietic progenitors. Most international guidelines, including the European LeukemiaNet 2022 update, recommend testing a comprehensive set of genes, most within a 3- to 5-day period for optimal treatment decisions. Next-generation sequencing gene panels are essential for identifying genetic alterations, risk stratification, and determining targeted therapies for myeloid malignancies. This study describes the analytical validation of the Oncomine Myeloid Assay GX v2 (Myeloid GX v2) in combination with the Ion Torrent Genexus System using commercial controls, 16 variant-negative samples, and 130 clinical samples of myeloid neoplasms. The Myeloid GX v2 panel detected single nucleotide variants (SNVs), insertions/deletions (indels) (allele frequency >5%), and gene fusions (minimum 11 fusion copies/µL) in synthetic controls with a sensitivity of 100%. Specificity for detection of SNVs, indels, or fusions in 16 variant-negative samples was 100%. Sensitivity for detection of SNVs, indels, and gene fusions in 130 clinical samples was 99%, 97%, and 100%, respectively. Overall precision was 100% for SNVs, 96% for indels, and 100% for fusions. The average turnaround time from nucleic acid extraction to results was 2 days. The Myeloid GX v2 panel is highly accurate and reproducible for the detection of SNVs, indels, and gene fusions in myeloid neoplasms. The ability to deliver clinically relevant results in a short time is key to providing personalized treatments.

BCR-ABL1 p210 screening for chronic myeloid leukemia in patients with peripheral blood cytoses.

INTRODUCTION: Chronic myeloid leukemia (CML) usually presents with leukocytosis with neutrophilia, left shift, and basophilia. Documentation of the BCR-ABL1 fusion is required for diagnosis, and this is often achieved via p210 BCR-ABL1 real-time polymerase chain reaction (RT-PCR). METHODS: Patients undergoing first-time testing for p210 BCR-ABL1 at our institution were retrospectively identified. The medical record was reviewed, and the patient age, sex, clinical indication for testing, and concurrent CBC with differential were identified for 518 patients. BCR-ABL1 p210 testing had been performed using a laboratory-developed quantitative RT-PCR assay. Statistical analysis of the results was performed using an unpaired t test, and P values of <.05 were considered statistically significant. RESULTS: Twenty-four patients received a new diagnosis of CML (4.6%). As compared to patients with a negative PCR, these patients were more likely to have a markedly elevated white blood cell count (WBC), neutrophilia, and a mild anemia. Ninety-two percent (22/24) of new CML patients had a WBC ≥20 × 109 /L, and the two new CML patients with WBC <20 × 109 /L had basophilia in the peripheral blood. By contrast, 92% (449/490) of non-CML patients had a WBC <20 × 109 /L. CONCLUSION: The peripheral blood parameters of total WBC ≥20 × 109 /L and absolute basophil count can help guide the need for BCR-ABL1 PCR testing, which can lead to more judicious test utilization, decreased healthcare costs, and decreased false positives, while keeping a high sensitivity for CML. This study also underscores the importance of obtaining a complete differential in patients for whom CML is suspected.

Sudden Unexpected Death in a Child From Acute Myeloid Leukemia.

ABSTRACT: Acute myeloid leukemia can rarely cause sudden, unexpected death in children. Presentation may be non-specific and death may occur in children with no prior medical history. Herein we present the case of a previously healthy 2-year and 2 month-old White girl, who on autopsy, was found to have acute myeloid leukemia with KMT2A rearrangement extensively involving all major thoracic and abdominal organs. This case is presented to the forensic community to discuss the presentation and findings in sudden death caused by acute leukemia. The case highlights when acute leukemia should enter the differential as a potential cause of death, as well as potential resources available in the postmortem workup of acute leukemias.

PML-RARA Fusion Transcripts Detectable 8 Months prior to Promyelocytic Blast Crisis in Chronic Myeloid Leukemia.

Promyelocytic blast crisis arising from chronic myeloid leukemia (CML) is rare. We present a 40-year-old male who developed promyelocytic blast crisis 17 months after CML diagnosis, confirmed by the presence of the t(15;17) and t(9;22) translocations in the leukemic cells. Preserved nucleic acids from routine BCR-ABL1 testing provided a unique opportunity to evaluate clonal progression over time. Retrospective analysis demonstrated PML-RARA fusion transcripts were first detectable 8 months prior to blast crisis presentation. A review of 21 cases of promyelocytic blasts crisis published in the literature reveals a male predominance with earlier age at onset as compared to females. Interestingly, TKI therapy during chronic phase did not impact the time interval between diagnosis and promyelocytic blast crisis. Treatment with standard acute promyelocytic leukemia regimens provides more favorable outcomes with complete molecular remission. Although rare, it is important to consider a promyelocytic blast crisis when evaluating for transformation of CML due to its effective treatment with specific therapies.

Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates.

BACKGROUND: Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. METHODS: In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. RESULTS: Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. CONCLUSIONS: FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society.

Chronic Lymphocytic Leukemia with Translocation (2;14)(p16;q32): A Case Report and Review of the Literature.

We report the case of a young African American male with no significant past medical history presenting with low back and bilateral leg pain; presenting CBC and chemistries revealed elevated white blood cell count of 250,000, with anemia (Hb 6.8 g/dL) and thrombocytopenia (platelets 9 K/µL), and elevated LDH, 1008. Physical examination findings were notable for diffuse lymphadenopathy and lower extremity skin nodules. Interestingly the bone marrow biopsy revealed involvement by CLL/SLL with translocation (2;14)(p16;q32) and trisomy 12. The patient was treated with fludarabine-based chemotherapy and steroids for CLL-related ITP with excellent response. After three cycles of chemotherapy, all the enlarged lymph nodes and skin nodules disappeared, and patient had achieved complete hematologic response. In this paper we also reviewed the available literature of CLL patients with translocation (2;14).

Significance of MYD88 L265P Mutation Status in the Subclassification of Low-Grade B-Cell Lymphoma/Leukemia.

CONTEXT: Lymphoplasmacytic lymphoma (LPL), marginal zone lymphoma (MZL), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) are well-defined clinicopathologic entities. However, distinguishing LPL from MZL and from atypical cases of CLL can sometimes be difficult because of overlapping features. Recent studies have identified a recurrent L265P mutation in the MYD88 gene in most cases of LPL. Although this represents a promising diagnostic marker for LPL, the mutation is also reported in rare cases of MZL and CLL (as well as other types of B-cell lymphoma). Detection rates for this mutation have varied depending on the analytic methodology. OBJECTIVE: To assess the diagnostic utility of MYD88 L265P mutation in diagnosing low-grade B-cell lymphomas. DESIGN: We developed a novel pyrosequencing assay for the MYD88 L265P mutation and assessed its diagnostic utility in 317 cases of low-grade B-cell lymphoma (45 LPL [14%], 53 MZL [17%], and 219 CLL [69%]). We incorporated formal clinical and pathologic review of selected cases to ensure the most accurate diagnosis and subclassification. RESULTS: The MYD88 L265P mutation was identified in 43 cases of LPL (96%), including 3 nonimmunoglobulin-M LPL cases. In contrast, the mutation was present in only 2 cases of MZL (4%), and 5 cases of CLL (2%). Thus, pyrosequencing for the MYD88 L265P mutation demonstrates a high clinical sensitivity and specificity to distinguish LPL from MZL and CLL. CONCLUSIONS: This study confirms the strong association of the MYD88 L265P mutation with LPL, as well as the existence of rare cases of small B-cell lymphoma that complicate this association.

Concurrent t(4;11) and t(1:19) in a Pediatric Patient with B-Lymphoblastic Leukemia/Lymphoma (B-ALL): A Case Report and Review of the Literature.

We herein present the case of a pediatric patient with B-lymphoblastic leukemia (B-ALL) with MLL gene rearrangement associated with the t(4;11)(q21;q23). Complete remission was achieved with standard B-ALL-directed chemotherapy and whole brain radiation. The patient subsequently relapsed and cytogenetic assessment revealed an additional acquired t(1;19)(q23;p13) associated with the TCF3-PBX1 fusion. After reinduction chemotherapy with a relapse B-ALL protocol the patient achieved disease remission; however, he developed respiratory complications and died. This represents a unique case as these two translocations have never been described concurrently in pediatric acute leukemia patients.

CD34+ megakaryocytes (≥30%) are associated with megaloblastic anaemia and non-acute myeloid neoplasia.

AIMS: To evaluate the sensitivity and specificity of CD34 staining of megakaryocytes (MKs), in order to distinguish non-neoplastic and neoplastic bone marrows (BMs). METHODS AND RESULTS: Three hundred BMs (120 non-neoplastic and 180 neoplastic) were evaluated for percentage and intensity of CD34 staining of MKs. The selected non-neoplastic cases included anaemia, autoimmune conditions, immune thrombocytopenia (ITP), and staging BMs. The neoplastic cases included myelodysplastic syndromes and/or myeloproliferative neoplasms (MDS, MPN, MDS/MPN). Eight per cent of non-neoplastic (9/120) cases and 13% of neoplastic (24/180) cases showed ≥30% CD34+ MKs, and these were essentially restricted to cases of megaloblastic anaemia (MBA) and non-acute myeloid neoplasms. The finding of ≥30% CD34+ MKs did not distinguish between categories of non-acute myeloid neoplasms. MDS cases with ≥30% CD34+ MKs had lower platelet counts than cases with <30% (P = 0.03). CONCLUSION: In complex BM cases, the presence of ≥30% CD34+ MKs constitutes a potentially useful diagnostic tool with which to distinguish non-acute myeloid neoplasms and MBA from non-MBA reactive conditions, for minimal additional cost.