Molecular Techniques and Gene Mutations in Myelodysplastic Syndromes.

Sequencing technology, particularly next-generation sequencing, has highlighted the importance of gene mutations in myelodysplastic syndromes (MDSs). Mutations affecting DNA methylation, chromatin modification, RNA splicing, cohesin complex, and other pathways are present in most MDS cases and often have prognostic and clinical implications. Updated international diagnostic guidelines as well as the new International Prognostic Scoring System-Molecular incorporate molecular data into the diagnosis and prognostication of MDS. With whole-genome sequencing predicted to become the future standard of genetic evaluation, it is likely that MDS diagnosis and management will become increasingly personalized based on an individual's clinical and genomic profile.

Diagnosis and Classification of Myelodysplastic Syndromes with Mutated TP53.

The genetic underpinnings of myeloid neoplasms are becoming increasingly well understood. The accessibility to sequencing technology, in particular next-generation sequencing (NGS), has highlighted the importance of gene mutations in myelodysplastic syndromes (MDS) in conjunction with traditional cytogenetics. With the relatively recent influx of molecular information to complement known cytogenetic abnormalities, the diagnosis, classification, and prognosis of MDS and acute myeloid leukemia (AML) have been increasingly refined, which has also led to therapeutic advancements. It has been shown that TP53 mutations have a significant impact in cases of MDS, as well as AML, and have led to TP53-defined myeloid disease. TP53 mutations are also now incorporated into prognostic scoring systems, as patients have been shown to have aggressive disease and poor outcomes. With the increased understanding of the importance of TP53 disruption in myeloid neoplasia, it is likely that the critical role of TP53 will continue to be highlighted by an individual's disease classification and personalized therapeutic management.

Helicobacter pylori-negative mucosa-associated lymphoid tissue (MALT) lymphoma of the stomach: A clinicopathologic analysis.

OBJECTIVES: Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is historically associated with Helicobacter pylori (HP) infections in more than 80% of patients. However, the incidence of HP-negative MALT lymphoma has been increasing. The clinicopathologic features have not been well studied, and optimal management strategies remain unclear. METHODS: The pathology database was searched for primary gastric MALT lymphomas diagnosed from 2000 to 2017. The clinical data and the slides were reviewed. The cases were divided for analysis into those with a background of chronic gastritis with HP, chronic gastritis without HP, and without either a background of chronic gastritis or HP. RESULTS: Of 70 gastric MALT lymphoma cases identified, 26 (37% of total) had chronic gastritis and were positive for HP histologically (n = 23) or were HP positive by additional laboratory testing (n = 3). The remaining 44 (63% of total) cases were HP negative by histology. Within the HP-negative cases, 5 (11% of HP-negative cases) showed histologic gastritis while 39 (89% of HP-negative cases) did not have sufficient evidence of gastritis through review of slides (n = 18) or based on available pathology reports (n = 21). The HP-negative cases without gastritis had higher propensities to show a mass lesion on endoscopy compared with HP-positive cases (37.5% vs 11.1%, P = .02) at the initial diagnosis. The immunophenotype and rate of positive B-cell gene rearrangement were not significantly different between the 2 groups. While all HP-positive patients received antibiotics for HP eradication, treatment in the HP-negative group varied among antibiotics, radiation, rituximab, or chemotherapy. Among HP-negative patients with available follow-up, 13 (39%) showed disease recurrence, similar to the recurrence rate in HP-positive patients; however, no individual from either group has died of the disease thus far. CONCLUSIONS: The incidence of HP-negative MALT lymphoma is increasing, and in our practice, it is currently more common than HP-associated MALT lymphomas. The pathophysiology of HP-negative MALT lymphoma without chronic gastritis remains unclear. Follow-up data in our study suggest that the prognosis of these cases is excellent despite varied management modalities.

Molecular findings in myeloid neoplasms.

The proliferation of new molecular technologies in recent years has greatly advanced our knowledge of the genetics that underlie hematologic cancers. Particularly, with the advent and wide-implementation of next-generation sequencing (NGS), a host of somatic (and some germline) gene mutations have been identified as significant in the classification, prognostication, and treatment of the spectrum of myeloid neoplasms. These driver and disease modifier mutations now play a prominent role in the updated international diagnostic guidelines of acute myeloid leukemia (AML), myelodysplastic syndromes/neoplasms (MDS), and myeloproliferative neoplasms (MPN). As high-throughput technologies such as NGS increasingly become standard in the genetic evaluation of myeloid disorders, it is critical that clinicians understand the clinical relevance of these mutations in order to further personalize patient care. In this review we discuss some of the most essential somatic and cytogenetic findings.

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.

Optimizing Donor Chimerism Threshold for Next-Generation Sequencing Monitoring of Measurable Residual Disease Post-Allogeneic Stem Cell Transplantation for Myeloid Neoplasms.

Next-generation sequencing (NGS) is used to monitor genetically measurable residual disease (gMRD) following allogeneic stem cell transplantation (aSCT). It is unknown whether an upper limit of chimerism exists such that gMRD NGS testing can be safely forgone. We reviewed 61 patients with acute myeloid leukemia and 24 patients with myelodysplastic syndrome who had at least 1 NGS panel before and after aSCT between 2016 and 2020. Donor chimerism was quantified. Logistic regression characterized which factors predicted gMRD. Receiver operating characteristic (ROC) curves were used to determine the optimal chimerism threshold for which gMRD would not be detected. Data from an additional 22 patients with follow-up NGS testing in 2022 were also analyzed to validate our proposed threshold. A: s expected, donor chimerism was a significant predictor of gMRD (odds ratio, .38; 95% confidence interval, .10 to .62; P = .02). Age, sex, conditioning regimen, presence of a related donor, and diagnosis were not associated with gMRD. A chimerism threshold of 92.5% optimized sensitivity (97.7%) and specificity (95.4%) such that values >92.5% strongly predicted the absence of gMRD (area under the ROC curve [AUC], .986). The validation cohort demonstrated similarly strong predictive capability (AUC, .974) with appropriate sensitivity (100%) and specificity (90.9%). NGS monitoring of gMRD is redundant at chimerism values greater than a more conservative threshold of 92.5% after aSCT.

Quantitative Risk for Single-Positive Lupus Anticoagulant Results With Different Anticoagulants.

OBJECTIVES: Clinical experts recommend against testing for lupus anticoagulant (LAC) during anticoagulation. METHODS: We quantitated the risk of a single-positive dilute Russell viper venom time (dRVVT) result or partial thromboplastin time-based phospholipid neutralization (PN) result on anticoagulation. RESULTS: Any anticoagulation led to a fourfold greater likelihood of single-positive results, primarily by rivaroxaban (odds ratio [OR] = 8.6) and warfarin (OR = 6.6), resulting in a positive dRVVT test with a normal PN test. Heparin and apixaban were twofold more likely to show single-positive results, but enoxaparin did not show significant single positivity. CONCLUSIONS: Our results quantitatively support experts' avoidance of LAC testing during anticoagulation.

An interactive e-learning module on peripheral blood smear analysis is an effective option for teaching pathology trainees.

OBJECTIVES: This study compares the effectiveness of an interactive e-learning module with a traditional text-based method for teaching peripheral blood smear analysis. METHODS: Pathology trainees at Accreditation Council for Graduate Medical Education residency programs were asked to participate. Participants completed a multiple-choice test on peripheral blood smear findings. Trainees were randomized into completing an e-learning module or a PDF reading exercise with the same educational content. Respondents rated their experience and completed a postintervention test composed of the same questions. RESULTS: In total, 28 participants completed the study; 21 improved their score in the posttest (mean, 21.6 correct answers) compared with the pretest (19.8; P < .001). This improvement was seen in both the PDF (n = 19) and interactive (n = 9) groups, with no difference in performance between the 2 groups. Trainees with less clinical hematopathology experience showed a trend of having the largest performance improvement. Most participants completed the exercise within 1 hour, rated the exercise as easy to navigate, were engaged, and reported learning new information about peripheral blood smear analysis. All participants indicated that they would likely complete a similar exercise in the future. CONCLUSIONS: This study suggests that e-learning is an effective tool for hematopathology education and equivalent to traditional narrative-based methods. This module could easily be incorporated into a curriculum.

Automated analysers underestimate atypical basophil count in myeloid neoplasms.

INTRODUCTION: Recent data suggest basophils can adopt an atypical appearance in myeloid disorders including myeloproliferative neoplasms (MPNs) and myeloproliferative/myelodysplastic disease. We hypothesized that automated analysers may not accurately quantitate basophils in myeloid neoplasms based on scatter properties. This study examined basophil counts and properties in myeloid disorders by automated cell analyser, manual differential, and flow cytometry. METHODS: Patients with myeloid neoplasms and control patients with no myeloid disorder diagnosis at a tertiary care centre were identified. Basophil percentage was compared for automated analyser counts (Sysmex XN9000), manual differential, and flow cytometry. Basophil scatter properties in MPNs were examined using flow cytometry. RESULTS: Thirty-one patients with myeloid neoplasms were included: 58% were male, mean age was 70.2 (±20.7) years, 32% had a diagnosis of chronic myeloid leukaemia with the remaining patients divided among various other forms of myeloid disease (including: essential thrombocythemia, polycythemia vera, unclassifiable MPN, myelodysplastic syndromes). For these patients, mean basophil percentage by automated analyser was significantly lower than manual differential (2.7 ± 2.9 vs. 7.1 ± 4.6, respectively, p < 0.001). No significant difference was found between automated versus manual differential for basophils in control subjects (p = 0.373). For myeloid neoplasm patients, mean basophil percentage was not significantly different between manual differential and flow cytometry (p = 0.116); mean basophil percentage by automated analyser was significantly lower than flow cytometry (2.7 ± 2.9 vs. 5.3 ± 3.7, respectively, p = 0.003). CONCLUSION: Automated analysers underestimate basophil counts in patients with myeloid neoplasms. Manual differential and flow cytometry are recommended for more accurate quantitation and characterization of aberrant basophils.

Genetic Landscape of Myeloproliferative Neoplasms with an Emphasis on Molecular Diagnostic Laboratory Testing.

Chronic myeloproliferative neoplasms (MPNs) are hematopoietic stem cell neoplasms with driver events including the BCR-ABL1 translocation leading to a diagnosis of chronic myeloid leukemia (CML), or somatic mutations in JAK2, CALR, or MPL resulting in Philadelphia-chromosome-negative MPNs with constitutive activation of the JAK-STAT signaling pathway. In the Philadelphia-chromosome-negative MPNs, modern sequencing panels have identified a vast molecular landscape including additional mutations in genes involved in splicing, signal transduction, DNA methylation, and chromatin modification such as ASXL1, SF3B1, SRSF2, and U2AF1. These additional mutations often influence prognosis in MPNs and therefore are increasingly important for risk stratification. This review focuses on the molecular alterations within the WHO classification of MPNs and laboratory testing used for diagnosis.

Laboratory evaluation and prognostication among adults and children with CEBPA-mutant acute myeloid leukemia.

CEBPA-mutant acute myeloid leukemia (AML) encompasses clinically and biologically distinct subtypes of AML in both adults and children. CEBPA-mutant AML may occur with monoallelic (moCEBPA) or biallelic (biCEBPA) mutations, which can be somatic or germline, with each entity impacting prognosis in unique ways. BiCEBPA AML is broadly associated with a favorable prognosis, but differences in the type and location of CEBPA mutations as well as the presence of additional leukemogenic mutations can lead to heterogeneity in survival. Concurrent FLT3-ITD mutations have a well-documented negative effect on survival in adult biCEBPA AML, whereas support for a negative prognostic effect of mutations in TET2, DNMT3A, WT1, CSF3R, ASXL1, and KIT is mixed. NPM1 and GATA2 mutations may have a positive prognostic impact. MoCEBPA AML has similar survival outcomes compared to AML with wild-type CEBPA, and risk stratification is determined by other cytogenetic and molecular findings. Germline CEBPA mutations may lead to familial biCEBPA AML after acquisition of second somatic CEBPA mutation, with variable penetrance and age. BiCEBPA AML in children is likely a favorable-risk diagnosis as it is in adults, but the role of a single CEBPA mutation and the impact of concurrent leukemogenic mutations are not clear in this population. Laboratory evaluation of the CEBPA gene includes PCR-based fragment-length analysis, Sanger sequencing, and next-generation sequencing. Phenotypic analysis using multiparameter flow cytometry can also provide additional data in evaluating CEBPA, helping to assess for the likelihood of mutation presence.

Clinical and Molecular Approach to Adult-Onset, Neoplastic Monocytosis.

PURPOSE OF REVIEW: In this review, we provide a comprehensive and contemporary understanding of malignant monocytosis and provide a framework by which the appropriate diagnosis with malignant monocytosis can be rendered. RECENT FINDINGS: Increasing data support the use of molecular data to refine the diagnostic approach to persistent monocytosis. The absence of a TET2, SRSF2, or ASXL1 mutation has ≥ 90% negative predictive value for a diagnosis of CMML. These data may also reliably differentiate chronic myelomonocytic leukemia, the malignancy that is most associated with mature monocytosis, from several other diseases that can be associated with typically a lesser degree of monocytosis. These include acute myelomonocytic leukemia, acute myeloid leukemia with monocytic differentiation, myelodysplastic syndromes, and myeloproliferative neoplasms driven by BCR-ABL1, PDGFRA, PDGFRB, or FGFR1 rearrangements or PCM1-JAK2 fusions among other rarer aberrations. The combination of monocyte partitioning with molecular data in patients with persistent monocytosis may increase the predictive power for the ultimate development of CMM but has not been prospectively validated. Many conditions, both benign and malignant, can be associated with an increase in mature circulating monocytes. After reasonably excluding a secondary or reactive monocytosis, there should be a concern for and investigation of malignant monocytosis, which includes hematopathologic review of blood and marrow tissues, flow cytometric analysis, and cytogenetic and molecular studies to arrive at an appropriate diagnosis.

Behind the scenes with basophils: an emerging therapeutic target.

Though basophils were originally viewed as redundant blood 'mast cells', the implementation of flow cytometry has established basophils as unique leukocytes with critical immunomodulatory functions. Basophils play an active role in allergic inflammation, autoimmunity, and hematological malignancies. They are distinguishable from other leukocytes by their characteristic metachromatic deep-purple cytoplasmic, round granules. Mature basophils are phenotypically characterized by surface expression of IL-3Rα (CD123); IL-3 drives basophil differentiation, degranulation, and synthesis of inflammatory mediators including type 2 cytokines. Basophil degranulation is the predominant source of histamine in peripheral blood, promoting allergic responses. Basophils serve as a bridge between innate and adaptive immunity by secreting IL-4 which supports eosinophil migration, monocyte differentiation into macrophages, B-cell activation, and CD4 T-cell differentiation into Th2 cells. Further, basophilia is a key phenomenon in myeloid neoplasms, especially chronic myeloid leukemia (CML) for which it is a diagnostic criterion. Increased circulating basophils, often with aberrant immunophenotype, have been detected in patients with CML and other myeloproliferative neoplasms (MPNs). The significance of basophils' immunoregulatory functions in malignant and non-malignant diseases is an active area of research. Ongoing and future research can inform the development of immunotherapies that target basophils to impact allergic, autoimmune, and malignant disease states. This review article aims to provide an overview of basophil biology, identification strategies, and roles and dysregulation in diseases.

Monocytic Acute Myeloid Leukemias with KM2TA Translocations to Chromosome 17q that May Clinically Mimic Acute Promyelocytic Leukemia.

OBJECTIVE: Acute promyelocytic leukemia (APL) with variant RARA translocation, eg, t(11;17), is not sensitive to all-trans retinoic acid and requires distinct chemotherapy. However, there are some leukemic entities that may mimic aspects of the clinical and/or laboratory picture of APL and cause confusion because of karyotype nomenclature. Therefore, recognition of such entities may be of therapeutic and prognostic significance. METHODS: We present 2 cases of acute myeloid leukemia (AML) with t(11;17) that were clinically concerning for APL based primarily on clinical presentation but were ultimately diagnosed as AML with monocytic differentiation. RESULTS: Both leukemias harbored KMT2A translocations, one located near but not involving RARA and the other with SEPT9. CONCLUSION: In leukemias that clinically and/or immunophenotypically mimic APL, identification of specific gene translocations can lead to the correct diagnosis and may carry therapeutic/prognostic implications.