Molecular techniques in haematopathology: what and how?

Here we review the 'what and how' of molecular techniques used in the context of haematopathological diagnostics of both lymphoid and myeloid neoplasms. Keeping in mind that the required resources for molecular testing are not universally available, we will not only discuss novel and emerging techniques that allow more high-throughput and sophisticated analyses of lymphoid and myeloid neoplasms, but also the more classical, low-cost alternatives and even some workarounds for molecular testing approaches. In this review we also address other key aspects around molecular techniques for haematopatholgy diagnostics, including preanalytics, data interpretation, and data management, bioinformatics, and interlaboratory precision and performance evaluation.

Clinical Validation of a Targeted Next-Generation Sequencing Panel for Lymphoid Malignancies.

Lymphoid malignancies are a heterogeneous group of hematological disorders characterized by a diverse range of morphologic, immunophenotypic, and clinical features. Next-generation sequencing (NGS) is increasingly being applied to delineate the complex nature of these malignancies and identify high-value biomarkers with diagnostic, prognostic, or therapeutic benefit. However, there are various challenges in using NGS routinely to characterize lymphoid malignancies, including pre-analytic issues, such as sequencing DNA from formalin-fixed, paraffin-embedded tissue, and optimizing the bioinformatic workflow for accurate variant calling and filtering. This study reports the clinical validation of a custom capture-based NGS panel to test for molecular markers in a range of lymphoproliferative diseases and histiocytic neoplasms. The fully validated clinical assay represents an accurate and sensitive tool for detection of single-nucleotide variants and small insertion/deletion events to facilitate the characterization and management of patients with hematologic cancers specifically of lymphoid origin.

Assessment of chimerism by next generation sequencing: A comparison to STR/qPCR methods.

Chimerism analysis is used to evaluate patients after allogeneic hematopoietic stem cell transplant (allo-HSCT) for engraftment and minimal measurable residual disease (MRD) monitoring. A combination of short-tandem repeat (STR) and quantitative polymerase chain reaction (qPCR) was required to achieve both sensitivity and accuracy in the patients with various chimerism statuses. In this study, an insertion/deletion-based multiplex chimerism assay by next generation sequencing (NGS) was evaluated using 5 simulated unrelated donor-recipient combinations from 10 volunteers. Median number of informative markers detected was 8 (range = 5 - 11). The limit of quantitation (LoQ) was determined to be 0.1 % recipient. Assay sample number/batch was 10-20 and total assay time was 19-31 h (manual labor = 2.1 h). Additionally, 50 peripheral blood samples from 5 allo-HSCT recipients (related: N = 4; unrelated: N = 1) were tested by NGS and STR/qPCR. Median number of informative markers detected was 7 (range = 4 - 12). Results from both assays demonstrated a strong correlation (Y = 0.9875X + 0.333; R2 = 0.9852), no significant assay bias (difference mean - 0.08), and 100 % concordant detection of percent recipient increase ≥ 0.1 % (indicator of increased relapse risk). NGS-based chimerism assay can support all allo-HSCT for engraftment and MRD monitoring and simplify clinical laboratory workflow compared to STR/qPCR.

Comprehensive morphologic characterization of bone marrow biopsy findings in a large cohort of patients with VEXAS syndrome: A single-institution longitudinal study of 111 bone marrow samples from 52 patients.

OBJECTIVES: VEXAS syndrome is an adult-onset autoinflammatory disease caused by a somatic pathogenic mutation in the UBA1 (ubiquitin-like modifier activating enzyme 1) gene. Patients present with rheumatologic manifestations and cytopenias and may have an increased predisposition to myelodysplastic syndrome (MDS) and plasma cell neoplasms. Prior studies have reported on the peripheral blood and bone marrow findings in patients with VEXAS syndrome. Due to the protean clinical presentation and lack of specificity of morphologic features (eg, vacuoles in early erythroid and granulocytic precursors), an optimal screening methodology to identify these patients in a timely fashion is desirable. METHODS: To further evaluate and describe the salient diagnostic morphologic features in VEXAS syndrome, we carried out a comprehensive study of the largest single-institution cohort to date. Diagnostic and follow-up bone marrow biopsy specimens from 52 male patients with molecularly identified VEXAS syndrome underwent central review. RESULTS: Cytopenias were common in all cases, primarily macrocytic anemia, monocytopenia, and thrombocytopenia. Bone marrow aspirate and biopsy were often hypercellular, with an increased myeloid/erythroid ratio, granulocytic hyperplasia with left shift, erythroid left shift, and megakaryocyte hyperplasia, which exhibited a range of striking morphologic findings. Distinctly vacuolated myeloid and erythroid precursors were seen in more than 95% of cases. CONCLUSIONS: Our data reveal potential novel diagnostic features, such as a high incidence of monocytopenia and distinct patterns of atypical megakaryopoiesis, that appear different from dysmegakaryopoiesis typically associated with MDS. In our experience, those findings are suggestive of VEXAS, in the appropriate clinical context.

Genetic landscape and clinical outcomes of patients with BCOR mutated myeloid neoplasms.

The BCL6-corepressor (BCOR) is a tumor-suppressor gene located on the short arm of chromosome X. Data are limited regarding factors predicting survival in BCOR-mutated (mBCOR) acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We evaluated 138 patients with mBCOR myeloid disorders, of which 36 (26.1%) had AML and 63 (45.6%) had MDS. Sixty-six (47.8%) patients had a normal karyotype while 18 (13%) patients had complex karyotype. BCOR-mutated MDS/AML were highly associated with RUNX1 and U2AF1 co-mutations. In contrast, TP53 mutation was infrequently seen with mBCOR MDS. Patients with an isolated BCOR mutation had similar survival compared to those with high-risk co-mutations by European LeukemiaNet (ELN) 2022 criteria (median OS 1.16 vs. 1.27 years, P=0.46). Complex karyotype adversely impacted survival among mBCOR AML/MDS (HR 4.12, P<0.001), while allogeneic stem cell transplant (alloSCT) improved survival (HR 0.38, P=0.04). However, RUNX1 co-mutation was associated with an increased risk of post-alloSCT relapse (HR 88.0, P=0.02), whereas melphalan-based conditioning was associated with a decreased relapse risk (HR 0.02, P=0.01). We conclude that mBCOR is a high-risk feature across MDS/AML, and that alloSCT improves survival in this population.

U2AF1 pathogenic variants in myeloid neoplasms and precursor states: distribution of co-mutations and prognostic heterogeneity.

We have previously recognized the genotypic and prognostic heterogeneity of U2AF1 mutations (MT) in myelofibrosis (MF) and myelodysplastic syndromes (MDS). In the current study, we considered 179 U2AF1-mutated patients with clonal cytopenia of undetermined significance (CCUS; n = 22), MDS (n = 108), MDS/acute myeloid leukemia (AML; n = 18) and AML (n = 31). U2AF1 variants included S34 (60%), Q157 (35%), and others (5%): corresponding mutational frequencies were 45%, 55%, and 0% in CCUS; 57%, 39%, and 4% in MDS; 61%, 33%, and 6% in MDS/AML; and 55%, 35% and 10% in AML (P = 0.17, 0.36 and 0.09), respectively. Concurrent mutations included ASXL1 (37%), BCOR (19%), RUNX1 (14%), TET2 (15%), DNMT3A (10%), NRAS/KRAS (8%), TP53 (8%), JAK2 (5.5%) and SETBP1 (5%). The two most frequent U2AF1 MT were S34F (n = 97) and Q157P (n = 46); concurrent MT were more likely to be seen with the latter (91% vs 74%; P = 0.01) and abnormal karyotype with the former (70% vs 62%; P = 0.05). U2AF1 S34F MT clustered with BCOR (P = 0.04) and Q157P MT with ASXL1 (P = 0.01) and TP53 (P = 0.03). The median overall survival (OS) in months was significantly worse in AML (14.2) vs MDS/AML (27.3) vs MDS (33.7; P = 0.001); the latter had similar OS with CCUS (30.0). In morphologically high-risk disease (n = 49), defined by ≥10% blood or bone marrow blasts (i.e., AML or MDS/AML), median OS was 14.2 with Q157P vs 37.1 months in the presence of S34F (P = 0.008); transplant-adjusted multivariable analysis confirmed the detrimental impact of Q157P (P = 0.01) on survival and also identified JAK2 MT as an additional risk factor (P = 0.02). OS was favorably affected by allogeneic hematopoietic stem cell transplantation (HR: 0.16, 95% CI; 0.04-0.61, P = 0.007). The current study defines the prevalence and co-mutational profiles of U2AF1 pathogenic variants in AML, MDS/AML, MDS, and CCUS, and suggests prognostic heterogeneity in patients with ≥10% blasts.

Real-world experience with ponatinib therapy in chronic phase chronic myeloid leukemia: impact of depth of response on survival and prior exposure to nilotinib on arterial occlusive events.

We surveyed the performance of ponatinib, as salvage therapy, in a real-world setting of chronic phase chronic myeloid leukemia (CML-CP). Among 55 consecutive patients (median age 49 years) with relapsed/refractory CML-CP, 35 (64%) had failed ≥3 tyrosine kinase inhibitors (TKIs), 35 (64%) were pre-treated with nilotinib, and 14 (28%) harbored ABL1T315I. At start of ponatinib (median dose 30 mg/day), 40 patients were already in complete hematologic (CHR), 4 in complete cytogenetic (CCyR), 3 in major molecular (MMR) remission, while 8 had not achieved CHR (NR). Ponatinib improved the depth of response in 13 (33%), 3 (75%), 2 (66%), and 4 (50%) patients with CHR, CCyR, MMR, and NR, respectively (p = 0.02). At a median follow-up of 42 months, 13 (23%) deaths, 5 (9%) blast transformations, and 25 (45%) allogeneic transplants were recorded. Five/10-year post-ponatinib survival was 77%/58% with no significant difference when patients were stratified by allogeneic transplant (p = 0.94), ponatinib-induced deeper response (p = 0.28), or a post-ponatinib ≥CCyR vs CHR remission state (p = 0.25). ABL1T315I was detrimental to survival (p = 0.04) but did not appear to affect response. Prior exposure to nilotinib was associated with higher risk of arterial occlusive events (AOEs; 11% vs 0%; age-adjusted p = 0.04). Ponatinib starting/maintenance dose (45 vs 15 mg/day) did not influence either treatment response or AOEs. Our observations support the use of a lower starting/maintenance dose for ponatinib in relapsed/refractory CML-CP but a survival advantage for deeper responses was not apparent and treatment might not overcome the detrimental impact of ABL1T315I on survival. The association between prior exposure to nilotinib and a higher risk of post-ponatinib AOEs requires further validation.

Follicular lymphoma and diffuse large B-cell lymphoma with BCL2 and IRF4 rearrangements in adult patients.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) with concurrent BCL2 and IRF4 rearrangements are rare. It is unclear whether such cases should be classified as large B- cell lymphoma with IRF4 rearrangement or FL/DLBCL-not otherwise specified. We identified 5 adult patients (FL, N = 3 and FL/DLBCL, N = 2) with concurrent BCL2 and IRF4 rearrangements. The median age at presentation was 77 years, and three patients presented with advanced stage disease. Both nodal and extranodal sites were involved and involvement was not limited to head and neck region. With a median follow-up of 18 months, 1 patient died and 4 patients were alive, including 3 who received chemotherapy and 1 who was observed. The neoplasms were histologically heterogeneous, including grade 2 and 3 FL and DLBCL. Four cases coexpressed CD10, BCL6, BCL2 and MUM1/IRF4. The Ki67 labelling index ranged from 20% to 95%. In 4 patients, the percentage of cells with BCL2 rearrangement was equal to or slightly greater than the cells harboring IRF4 rearrangement. Two cases underwent next generation sequencing tailored for lymphoid neoplasms. Both lacked mutations involving IRF4 and NF-kB pathway genes that are frequently detected in large B-cell lymphoma with IRF4 rearrangement, and one case showed DLBCL-EZH2 type mutations, including KMT2D and BCL2 mutations, similar to 2 previously reported DLBCL with BCL2 and IRF4 rearrangements. Adults with FL and FL/DLBCL with BCL2 and IRF4 rearrangements display clinicopathologic and mutational features more akin to FL and DLBCL and should not be characterized as large B-cell lymphoma with IRF4 rearrangement.

Malignant Histiocytosis Comprises a Phenotypic Spectrum That Parallels the Lineage Differentiation of Monocytes, Macrophages, Dendritic Cells, and Langerhans Cells.

Malignant histiocytoses (MHs), or the 'M group' of the Histiocyte Society classification, are characterized by neoplastic histiocytes with large pleomorphic nuclei. MH encompasses the diagnoses of histiocytic sarcoma, interdigitating dendritic cell sarcoma, and Langerhans cell sarcoma. We aimed to define the phenotypic spectrum of MH and examine the genotypic features across this spectrum. Using immunohistochemistry, we arranged the 22 cases into 4 subtypes that correspond to the lines of differentiation from monocytic and dendritic cell precursors as follows: (1) macrophage (n = 5): CD68+, CD163+, CD14+, and Factor 13a+; (2) monocyte-macrophage (n = 5): CD68+, CD163+, CD14+, S100+, and OCT2+; (3) dendritic cell (n = 6): CD68+, CD11c+, S100+, lysozyme+, ZBTB46+, and CD1a/langerin < 5%; and (4) Langerhans cell (n = 6): CD68+, CD11c+, S100+, ZBTB46+, CD1a+, and langerin+. The phenotypic subtypes align with those seen in low-grade histiocytic neoplasms as follows: MH-macrophage type correlates with Erdheim-Chester disease phenotype; MH-monocyte-macrophage type with Rosai-Dorfman disease phenotype, and MH-Langerhans cell type with Langerhans cell histiocytosis. Activating mutations in MAPK-pathway genes were identified in 80% of MH cases; 29% had mutations in the PI3k-AKT-mTOR pathway and 59% had mutations in epigenetic modulating genes. Strong expression of cyclin D1 was present in all cases, whereas p-ERK and p-AKT were not uniformly expressed. Eight of 22 (36%) MH cases were proven to be clonally related to a prior B-cell lymphoma. Defining the phenotypic spectrum of MH provides a guide to diagnosis and allows further exploration into the potential biological and clinical significance.

Clinical and molecular correlates of somatic and germline DDX41 variants in patients and families with myeloid neoplasms.

The diagnosis of germline predisposition to myeloid neoplasms (MN) secondary to DDX41 variants is currently hindered by the long latency period, variable family histories and the frequent occurrence of DDX41 variants of uncertain significance (VUS). We reviewed 4,524 consecutive patients who underwent targeted sequencing for suspected or known MN and analyzed the clinical impact and relevance of DDX41VUS in comparison to DDX41path variants. Among 107 patients (44 [0.9%] DDX41path and 63 DDX41VUS [1.4%; 11 patients with both DDX41path and DDX41VUS]), we identified 17 unique DDX41path and 45 DDX41VUS variants: 24 (23%) and 77 (72%) patients had proven and presumed germline DDX41 variants, respectively. The median age was similar between DDX41path and DDX41VUS (66 vs. 62 years; P=0.41). The median variant allele frequency (VAF) (47% vs. 48%; P=0.62), frequency of somatic myeloid co-mutations (34% vs 25%; P= 0.28), cytogenetic abnormalities (16% vs. 12%; P=>0.99) and family history of hematological malignancies (20% vs. 33%; P=0.59) were comparable between the two groups. Time to treatment in months (1.53 vs. 0.3; P=0.16) and proportion of patients progressing to acute myeloid leukemia (14% vs. 11%; P=0.68), were similar. The median overall survival in patients with high-risk myelodysplastic syndrome/acute myloid leukemia was 63.4 and 55.7 months in the context of DDX41path and DDX41VUS, respectively (P=0.93). Comparable molecular profiles and clinical outcomes among DDX41path and DDX41VUS patients highlights the need for a comprehensive DDX41 variant interrogation/classification system, to improve surveillance and management strategies in patients and families with germline DDX41 predisposition syndromes.

MICon Contamination Detection Workflow for Next-Generation Sequencing Laboratories Using Microhaplotype Loci and Supervised Learning.

Innovation in sequencing instrumentation is increasing the per-batch data volumes and decreasing the per-base costs. Multiplexed chemistry protocols after the addition of index tags have further contributed to efficient and cost-effective sequencer utilization. With these pooled processing strategies, however, comes an increased risk of sample contamination. Sample contamination poses a risk of missing critical variants in a patient sample or wrongly reporting variants derived from the contaminant, which are particularly relevant issues in oncology specimen testing in which low variant allele frequencies have clinical relevance. Small custom-targeted next-generation sequencing (NGS) panels yield limited variants and pose challenges in delineating true somatic variants versus contamination calls. A number of popular contamination identification tools have the ability to perform well in whole-genome/exome sequencing data; however, in smaller gene panels, there are fewer variant candidates for the tools to perform accurately. To prevent clinical reporting of potentially contaminated samples in small next-generation sequencing panels, we have developed MICon (Microhaplotype Contamination detection), a novel contamination detection model that uses microhaplotype site variant allele frequencies. In a heterogeneous hold-out test cohort of 210 samples, the model displayed state-of-the-art performance with an area under the receiver-operating characteristic curve of 0.995.

The clinical and molecular spectrum of ETV6 mutated myeloid neoplasms.

ETV6 mutations are rare but recurrent somatic events in myeloid neoplasms and are negatively prognostic in myelodysplastic syndrome. We set out to examine the clinical and molecular characteristics of patients undergoing investigation for myeloid neoplasms, found to have deleterious ETV6 mutations. ETV6 mutations occurred in 33 of 5793 (0.6%) cases investigated and predominantly in high-risk disease entities including MDS with increased blasts, primary myelofibrosis and AML, myelodysplasia-related. In three cases, isolated iso (17q) karyotype was concurrently detected, an otherwise rare karyotype in myeloid neoplasms. ETV6 mutations were frequently subclonal and never occurred as an isolated abnormality with ASXL1 (n = 22, 75%), SRSF2 (n = 14, 42%) and SETBP1 (n = 11, 33%) the predominant co-mutations. Restricting to patients with MDS, higher rates of ASXL1, SETBP1, RUNX1 and U2AF1 mutations occurred in ETV6 mutated cases, relative to a consecutive control cohort with wild-type ETV6. The median OS of the cohort was 17.5 months. This report highlights the clinical and molecular associations of somatic ETV6 mutations in myeloid neoplasms, suggests their occurrence as a later event, and proposes further translational research questions for their role in myeloid neoplasia.