The genetic landscape of germline DDX41 variants predisposing to myeloid neoplasms.

Germline DDX41 variants are the most common mutations predisposing to acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) in adults, but the causal variant (CV) landscape and clinical spectrum of hematologic malignancies (HMs) remain unexplored. Here, we analyzed the genomic profiles of 176 patients with HM carrying 82 distinct presumably germline DDX41 variants among a group of 9821 unrelated patients. Using our proposed DDX41-specific variant classification, we identified features distinguishing 116 patients with HM with CV from 60 patients with HM with variant of uncertain significance (VUS): an older age (median 69 years), male predominance (74% in CV vs 60% in VUS, P = .03), frequent concurrent somatic DDX41 variants (79% in CV vs 5% in VUS, P < .0001), a lower somatic mutation burden (1.4 ± 0.1 in CV vs 2.9 ± 0.04 in VUS, P = .012), near exclusion of canonical recurrent genetic abnormalities including mutations in NPM1, CEBPA, and FLT3 in AML, and favorable overall survival (OS) in patients with AML/MDS. This superior OS was determined independent of blast count, abnormal karyotypes, and concurrent variants, including TP53 in patients with AML/MDS, regardless of patient's sex, age, or specific germline CV, suggesting that germline DDX41 variants define a distinct clinical entity. Furthermore, unrelated patients with myeloproliferative neoplasm and B-cell lymphoma were linked by DDX41 CV, thus expanding the known disease spectrum. This study outlines the CV landscape, expands the phenotypic spectrum in unrelated DDX41-mutated patients, and underscores the urgent need for gene-specific diagnostic and clinical management guidelines.

JAK2 R683S Mutation Resulting in Dual Diagnoses of Chronic Eosinophilic Leukemia and Myelodysplastic/Myeloproliferative Overlap Syndrome.

A 66-year-old male presented with hypereosinophilia, thrombocytosis, extensive thrombosis refractory to direct oral anticoagulant therapy, and evidence of end-organ damage, including rash, splenic infarcts, and pulmonary infiltrates. Bone marrow biopsy revealed myeloid malignancy consistent with both chronic eosinophilic leukemia and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with SF3B1 mutation and thrombocytosis. Next-generation sequencing of the patient's eosinophils and neutrophil compartments revealed pathologic variants in EZH2 and SF3B1 in addition to a noncanonical JAK2 R683S mutation that has not been previously described in myeloproliferative disorders or other chronic myeloid neoplasms. These mutations were not present in the patient's lymphoid cell fraction, suggesting that the hematopoietic malignancy arose in a myeloid-committed progenitor cell. Based on this case and previous work from our group, we propose that noncanonical JAK2 mutations may permit signal transduction that biases toward eosinophilic differentiation in chronic myeloid neoplasms. Although the patient's blood counts initially responded to ruxolitinib and hydroxyurea, the response was not durable. Early referral for allogenic bone marrow transplant appears necessary to prevent long-term complications and disease progression in myeloid neoplasms with clonal hypereosinophilia driven by noncanonical JAK2 mutations.

Clinical utility of targeted next-generation sequencing panel in routine diagnosis of hereditary hemolytic anemia: A national reference laboratory experience.

INTRODUCTION: Hereditary hemolytic anemias (HHA) comprise a heterogeneous group of disorders resulting from defective red blood cell (RBC) cytoskeleton, RBC enzyme deficiencies, and hemoglobin (Hb) synthesis disorders such as thalassemia or sideroblastic anemia. MATERIALS AND METHODS: Our hemolytic anemia diagnostic next-generation sequencing (NGS) panel includes 28 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The panel covers the complete coding region of these genes, splice junctions, and, wherever appropriate, deep intronic or regulatory regions are also included. Four hundred fifty-six patients with unexplained hemolytic anemia were evaluated using our NGS panel between 2015 and 2019. RESULTS: We identified pathogenic/likely pathogenic variants in 111/456 (24%) patients that were responsible for the disease phenotype (e.g., moderate to severe hemolytic anemia and hyperbilirubinemia). Approximately 40% of the mutations were novel. As expected, 45/456 (10%) patients were homozygous for the promoter polymorphism in the UGT1A1 gene, A(TA)7 TAA (UGT1A1*28). 8/45 homozygous UGT1A1*28 cases were associated with additional pathogenic mutations causing hemolytic anemia, likely exacerbating hyperbilirubinemia. The most common mutated genes were membrane cytoskeleton genes SPTA1, and SPTB, followed by PKLR. Complex interactions between SPTA1 low expression alleles, alpha-LELY and alpha-LEPRA alleles, and intragenic SPTA1 variants were associated with hereditary pyropoikilocytosis and autosomal recessive hereditary spherocytosis in 23/111 patients. CONCLUSIONS: Our results demonstrate that hemolytic anemia is underscored by complex molecular interactions of previously known and novel mutations in RBC cytoskeleton/enzyme genes, and therefore, NGS should be considered in all patients with clinically unexplained hemolytic anemia and in neonates with hyperbilirubinemia. Moreover, low expression alleles alpha-LELY and alpha-LEPRA should be included in all targeted HHA panels.

Near Complete Response to Trametinib Treatment in Histiocytic Sarcoma Harboring a Somatic KRAS Mutation.

Survival outcomes of patients with histiocytic neoplasms are poor, with no standard-of-care treatments available for these malignancies. Recent characterization of the genomic landscape of various histiocytic neoplasms have shown a predominance of activating driver mutations within the MAPK/ERK pathway (ie, BRAF, MEK, KRAS, MAPK, and NRAS). Subsequently, successful treatment of these malignancies with BRAF and MEK inhibitors has been reported. This report presents the first patient with histiocytic sarcoma harboring a somatic KRAS Q61H mutation who was subsequently treated to a near complete response with the MEK inhibitor trametinib. Due to patient preference, lack of standard of care treatments, and associated morbidity from head and neck dissection, initial disease reduction provided by trametinib therapy allowed for a less morbid resection. This case report highlights the utility of up-front next-generation sequencing and the efficacy of MEK inhibition in patients with histiocytic sarcoma harboring activating KRAS mutations.

Diagnostic Challenge and Clinical Dilemma: The Long Reach of Clonal Hematopoiesis.

BACKGROUND: Widespread application of massively parallel sequencing has resulted in recognition of clonal hematopoiesis in various clinical settings and on a relatively frequent basis. Somatic mutations occur in individuals with normal blood counts, and increase in frequency with age. The genes affected are the same genes that are commonly mutated in overt myeloid malignancies such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). This phenomenon is referred to as clonal hematopoiesis of indeterminate potential (CHIP). CONTENT: In this review, we explore the diagnostic and clinical implications of clonal hematopoiesis. In addition to CHIP, clonal hematopoiesis may be seen in patients with cytopenia who do not otherwise meet criteria for hematologic malignancy, a condition referred to as clonal cytopenia of undetermined significance (CCUS). Distinguishing CHIP and CCUS from overt myeloid neoplasm is a challenge to diagnosticians due to the overlapping mutational landscape observed in these conditions. We describe helpful laboratory and clinical features in making this distinction. CHIP confers a risk of progression to overt hematologic malignancy similar to other premalignant states. CHIP is also associated with a proinflammatory state with multisystem implications and increased mortality risk due to cardiovascular events. The current approach to follow up and management of patients with clonal hematopoiesis is described. SUMMARY: Nuanced understanding of clonal hematopoiesis is essential for diagnosis and clinical management of patients with hematologic conditions. Further data are needed to more accurately predict the natural history and guide management of these patients with respect to both malignant progression as well as nonhematologic sequelae.

KLF1/EKLF expression in acute leukemia is correlated with chromosomal abnormalities.

KLF1 (EKLF) is a master regulator of erythropoiesis and controls expression of a wide array of target genes. We interrogated human tissue microarray samples via immunohistological analysis to address whether levels of KLF1 protein are associated with leukemia. We have made the unexpected findings that higher KLF1 levels are correlated with cells containing abnormal chromosomes, and that high KLF1 expression is not limited to acute myeloid leukemia (AML) associated with erythroid/megakaryoblastic differentiation. Expression of KLF1 is associated with poor survival. Further analyses reveal that KLF1 directly regulates a number of genes that play a role in chromosomal integrity. Together these results suggest that monitoring KLF1 levels may provide a new marker for risk stratification and prognosis in patients with AML.

Concurrent detection of targeted copy number variants and mutations using a myeloid malignancy next generation sequencing panel allows comprehensive genetic analysis using a single testing strategy.

Currently, comprehensive genetic testing of myeloid malignancies requires multiple testing strategies with high costs. Somatic mutations can be detected by next generation sequencing (NGS) but copy number variants (CNVs) require cytogenetic methods including karyotyping, fluorescence in situ hybidization and microarray. Here, we evaluated a new method for CNV detection using read depth data derived from a targeted NGS mutation panel. In a cohort of 270 samples, we detected pathogenic mutations in 208 samples and targeted CNVs in 68 cases. The most frequent CNVs were 7q deletion including LUC7L2 and EZH2, TP53 deletion, ETV6 deletion, gain of RAD21 on 8q, and 5q deletion, including NSD1 and NPM1. We were also able to detect exon-level duplications, including so-called KMT2A (MLL) partial tandem duplication, in 9 cases. In the 63 cases that were negative for mutations, targeted CNVs were observed in 4 cases. Targeted CNV detection by NGS had very high concordance with single nucleotide polymorphism microarray, the current gold standard. We found that ETV6 deletion was strongly associated with TP53 alterations and 7q deletion was associated with mutations in TP53, KRAS and IDH1. This proof-of-concept study demonstrates the feasibility of using the same NGS data to simultaneously detect both somatic mutations and targeted CNVs.

Clinical utility of next-generation sequencing in the diagnosis of hereditary haemolytic anaemias.

Hereditary haemolytic anaemias are genetically and phenotypically heterogeneous disorders characterized by increased red cell destruction, with consequences ranging from innocuous to severe life-threatening anaemia. Diagnostic laboratories endeavour to assist clinicians reach the exact patient diagnosis by using tests principally based on morphological and biochemical techniques. However, these routine studies may be inconclusive, particularly in newborn infants and when transfusions have recently been administered. Large numbers and size of the potentially involved genes also impose a practical challenge for molecular diagnosis using routine sequencing approaches. To overcome these diagnostic shortcomings, we have utilized next-generation sequencing to provide a high-throughput, highly sensitive assay. We developed a panel interrogating 28 genes encoding cytoskeletal proteins and enzymes with sequencing coverage of the coding regions, splice site junctions, deep intronic and regulatory regions. We then evaluated 19 samples, including infants with unexplained extreme hyperbilirubinaemia and patients with transfusion-dependent haemolytic anaemia. Where possible, inheritance patterns of pathogenic mutations were determined by sequencing of immediate relatives. We conclude that this next-generation sequencing panel could be a cost-effective approach to molecular diagnosis of hereditary haemolytic anaemia, especially when the family history is uninformative or when routine laboratory testing fails to identify the causative haemolytic process.

Optimized immunohistochemical panel to differentiate myeloid sarcoma from blastic plasmacytoid dendritic cell neoplasm.

Myeloid sarcoma (MS) and blastic plasmacytoid dendritic cell neoplasm (BPDCN) can be difficult to distinguish morphologically, even with the use of extensive immunohistochemical studies. Three new research markers, myxovirus A (MxA), CLA/CD162, and CD303/BDCA-2, have been reported to be positive in BPDCN, but their clinical utility has never been tested. We compared these markers to other antibodies that have been used traditionally to distinguish MS from BPDCN to assess the utility of these newer antibodies in differential diagnosis. Formalin-fixed, paraffin-embedded tissue sections of 23 MS and 17 BPDCN cases were assessed using immunohistochemical analysis for CD4, CD14, CD33, CD43, CD56, CD68, CD123, CD163, myeloperoxidase, lysozyme, terminal deoxynucleotidyl transferase (TdT), T-cell leukemia 1 (TCL-1), MxA, cutaneous lymphocyte-associated antigen (CLA)/CD162, and blood dendritic cell antigen 2 (BDCA2)/CD303. We identified antibodies with a high predictive value of ≥ 90% and used these markers to develop an approach to classification using specific staining criteria. Diagnostic classification criteria were based on staining patterns of one or more of the seven markers. BPDCN was associated with positive staining for CD56, TdT, or TCL1, or negative staining for lysozyme. MS was associated with positive staining for lysozyme or myeloperoxidase, or negative staining for CD56, CD123, myxovirus, or TCL1. The immunohistochemical staining patterns observed using a panel that includes MPO, CD56, CD123, TCL1, TdT, and MxA, are predictive of MS or BPDCN. In this study, neither CD162 nor CD303 had good predictive value in distinguishing MS from BPDCN.

LMO2 (LIM domain only 2) is expressed in a subset of acute myeloid leukaemia and correlates with normal karyotype.

AIM: LMO2 is a transcription factor that plays a key role in haematopoiesis. Expression of LMO2 has been demonstrated in germinal centre B cells, various B cell lymphomas and T lymphoblastic lymphoma/leukaemia (T-ALL), but has not been studied extensively in acute myeloid leukaemia (AML). METHODS: We studied LMO2 expression by immunohistochemistry in biopsies from a cohort of AML patients (n = 196) and correlated it with established prognostic factors such as age, bone marrow morphology and cytogenetic findings. RESULTS: Forty per cent (79 of 196) of the samples from AML patients showed moderate/strong expression of LMO2 protein. LMO2 expression showed a significant positive correlation with normal cytogenetics (65% versus 24%, P < 0.0001) and a moderately negative correlation with complex karyotype [rs (98) = -0.218, P < 0.002]. AML associated with core binding factor [(t(8;21)/inv(16)/t(16;16)] had low LMO2 expression compared to diploid karyotype (29% versus 65%; P = 0.013). Expression of LMO2 protein exhibited an insignificant association with age (P = 0.197). Lower expression of LMO2 protein was noted in AML associated with myelodysplasia-related changes, compared to AML subtypes based on FAB classification (M0-M7) (21% versus 44%, P = 0.0187). CONCLUSIONS: LMO2 is expressed in a subset of AML patients and is associated with normal karyotype, which is different from T-ALL, where specific translocation (11p13) mediates protein expression.

Real world predictors of response and 24-month survival in high-grade TP53-mutated myeloid neoplasms.

Current therapies for high-grade TP53-mutated myeloid neoplasms (≥10% blasts) do not offer a meaningful survival benefit except allogeneic stem cell transplantation in the minority who achieve a complete response to first line therapy (CR1). To identify reliable pre-therapy predictors of complete response to first-line therapy (CR1) and outcomes, we assembled a cohort of 242 individuals with TP53-mutated myeloid neoplasms and ≥10% blasts with well-annotated clinical, molecular and pathology data. Key outcomes examined were CR1 & 24-month survival (OS24). In this elderly cohort (median age 68.2 years) with 74.0% receiving frontline non-intensive regimens (hypomethylating agents +/- venetoclax), the overall cohort CR1 rate was 25.6% (50/195). We additionally identified several pre-therapy factors predictive of inferior CR1 including male gender (P = 0.026), ≥2 autosomal monosomies (P < 0.001), -17/17p (P = 0.011), multi-hit TP53 allelic state (P < 0.001) and CUX1 co-alterations (P = 0.010). In univariable analysis of the entire cohort, inferior OS24 was predicated by ≥2 monosomies (P = 0.004), TP53 VAF > 25% (P = 0.002), TP53 splice junction mutations (P = 0.007) and antecedent treated myeloid neoplasm (P = 0.001). In addition, mutations/deletions in CUX1, U2AF1, EZH2, TET2, CBL, or KRAS ('EPI6' signature) predicted inferior OS24 (HR = 2.0 [1.5-2.8]; P < 0.0001). In a subgroup analysis of HMA +/-Ven treated individuals (N = 144), TP53 VAF and monosomies did not impact OS24. A risk score for HMA +/-Ven treated individuals incorporating three pre-therapy predictors including TP53 splice junction mutations, EPI6 and antecedent treated myeloid neoplasm stratified 3 prognostic distinct groups: intermediate, intermediate-poor, and poor with significantly different median (12.8, 6.0, 4.3 months) and 24-month (20.9%, 5.7%, 0.5%) survival (P < 0.0001). For the first time, in a seemingly monolithic high-risk cohort, our data identifies several baseline factors that predict response and 24-month survival.

Genomic Data Heterogeneity across Molecular Diagnostic Laboratories: A Real-World Connect Myeloid Disease Registry Perspective on Variabilities in Genomic Assay Methodology and Reporting.

Genomic data variability from laboratory reports can impact clinical decisions and population-level analyses; however, the extent of this variability and the impact on the data's value are not well characterized. This pilot study used anonymized genetic and genomic test reports from the Connect Myeloid Disease Registry (NCT01688011), a multicenter, prospective, observational cohort study of patients with newly diagnosed myelodysplastic syndromes, acute myeloid leukemia, or idiopathic cytopenia of undetermined significance, to analyze laboratory test variabilities and limitations. Results for 56 randomly selected patients enrolled in the Registry were independently extracted and evaluated (data cutoff, January 2020). Ninety-five reports describing 113 assay results from these 56 patients were analyzed for discrepancies. Almost all assay results [101 (89%)] identified the sequencing technology applied, and 94 (83%) described the test limitations; 95 (84%) described the limits of detection, but none described the limit of blank for detecting false positives. RNA transcript identifiers were not provided for 20 (43%) variants analyzed by next-generation sequencing and reported by the same laboratory. Of 42 variants with variant allele frequencies ≥30%, 16 (38%) of the variants did not have report text indicating that the variants might be germline. Variabilities and lack of standardization present challenges for incorporating this information into clinical care and render data collation ineffective and unreliable for large-scale use in centralized databases for therapeutic discovery.

Clinicopathologic characteristics of myeloproliferative neoplasms with JAK2 exon 12 mutation.

The presence of JAK2 exon 12 mutation was included by the 2016 World Health Organization (WHO) Classification as one of the major criteria for diagnosing polycythemia vera (PV). Few studies have evaluated the clinical presentation and bone marrow morphology of these patients and it is unclear if these patients fulfill the newly published criteria of 5th edition WHO or The International Consensus Classification (ICC) criteria for PV. Forty-three patients with JAK2 exon 12 mutations were identified from the files of 7 large academic institutions. Twenty patients had complete CBC and BM data at disease onset. Fourteen patients met the diagnostic criteria for PV and the remaining six patients were diagnosed as MPN-U. At diagnosis, 9/14 patients had normal WBC and platelet counts (isolated erythrocytosis/IE subset); while 5/14 had elevated WBC and/or platelets (polycythemic /P subset). We found that hemoglobin and hematocrit tended to be lower in the polycythemia group. Regardless of presentation (P vs IE), JAK2 deletion commonly occurred in amino acids 541-544 (62 %). MPN-U patients carried JAK2 exon 12 mutation, but did not fulfill the criteria for PV. Half of the patients had hemoglobin/hematocrit below the diagnostic threshold for PV, but showed increased red blood cell count with low mean corpuscular volume (56-60 fL). Three cases lacked evidence of bone marrow hypercellularity. In summary, the future diagnostic criteria for PV may require a modification to account for the variant CBC and BM findings in some patients with JAK2 exon 12 mutation.

Transplantation Referral Patterns for Patients with Newly Diagnosed Higher-Risk Myelodysplastic Syndromes and Acute Myeloid Leukemia at Academic and Community Sites in the Connect® Myeloid Disease Registry: Potential Barriers to Care.

Hematopoietic stem cell transplantation (HCT) is indicated for patients with higher-risk (HR) myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Age, performance status, patient frailty, comorbidities, and nonclinical factors (eg, cost, distance to site) are all recognized as important clinical factors that can influence HCT referral patterns and patient outcomes; however, the proportion of eligible patients referred for HCT in routine clinical practice is largely unknown. This study aimed to assess patterns of consideration for HCT among patients with HR-MDS and AML enrolled in the Connect® Myeloid Disease Registry at community/government (CO/GOV)- or academic (AC)-based sites, as well as to identify factors associated with rates of transplantation referral. We assessed patterns of consideration for and completion of HCT in patients with HR-MDS and AML enrolled between December 12, 2013, and March 6, 2020, in the Connect Myeloid Disease Registry at 164 CO/GOV and AC sites. Registry sites recorded whether patients were considered for transplantation at baseline and at each follow-up visit. The following answers were possible: "considered potentially eligible," "not considered potentially eligible," or "not assessed." Sites also recorded whether patients subsequently underwent HCT at each follow-up visit. Rates of consideration for HCT between CO/GOV and AC sites were compared using multivariable logistic regression analysis with covariates for age and comorbidity. Among the 778 patients with HR-MDS or AML enrolled in the Connect Myeloid Disease Registry, patients at CO/GOV sites were less likely to be considered potentially eligible for HCT than patients at AC sites (27.9% versus 43.9%; P < .0001). Multivariable logistic regression analysis with factors for age (<65 versus ≥65 years) and ACE-27 comorbidity grade (<2 versus ≥2) showed that patients at CO/GOV sites were significantly less likely than those at AC sites to be considered potentially eligible for HCT (odds ratio, 1.6, 95% confidence interval, 1.1 to 2.4; P = .0155). Among patients considered eligible for HCT, 45.1% (65 of 144) of those at CO/GOV sites and 35.7% (41 of 115) of those at AC sites underwent transplantation (P = .12). Approximately one-half of all patients at CO/GOV (50.1%) and AC (45.4%) sites were not considered potentially eligible for HCT; the most common reasons were age at CO/GOV sites (71.5%) and comorbidities at AC sites (52.1%). Across all sites, 17.4% of patients were reported as not assessed (and thus not considered) for HCT by their treating physician (20.7% at CO/GOV sites and 10.7% at AC sites; P = .0005). These findings suggest that many patients with HR-MDS and AML who may be candidates for HCT are not receiving assessment or consideration for transplantation in clinical practice. In addition, treatment at CO/GOV sites and age remain significant barriers to ensuring that all potentially eligible patients are assessed for HCT.

The immunophenotype of T-lymphoblastic lymphoma in children and adolescents: a Children's Oncology Group report.

T-lymphoblastic leukaemia (T-ALL) and T-lymphoblastic lymphoma (T-LBL) are neoplasms derived from immature lymphoid cells of T-cell lineage. These neoplasms are biologically similar, but significant differences may exist between the two given their clinical differences. Although ample data regarding the immunophenotypic characterization T-ALL are available, there is a paucity of such data in children and adolescents with T-LBL. We used flow cytometry and/or immunohistochemistry to characterize the immunophenotypic profile of 180 children and adolescents with newly diagnosed T-LBL enrolled in the Children's Oncology Group 5971 study. Multiple T-cell, B-cell, myeloid, and other markers were evaluated. We identified diagnostically useful immunophenotypic features of T-LBL as well as distinct immunophenotypic subgroups, although none of these was statistically related to event-free or overall survival in this retrospective analysis. Further studies of biologically and immunophenotypically distinct subgroups of T-LBL, such as the early T-cell precursor phenotype, are warranted.

AML with germline DDX41 variants is a clinicopathologically distinct entity with an indolent clinical course and favorable outcome.

Germline DDX41 variants in myeloid neoplasms (MNs) are not uncommon, and we explored the prevalence and characterized the clinical and pathologic features in a cohort of 3132 unrelated adult MN patients. By targeted next-generation sequencing, we identified 28 patients (20 men and 8 women) with pathogenic germline DDX41 variants who developed acute myeloid leukemia (AML), in which only 3 (11%) had a family history (FH) of MNs. A subacute clinical course of cytopenia (mean duration of 11.2 months, range 0-72 months) prior to the initial AML diagnosis was accompanied by a low blast count (median at 30%, range 20-70%) in hypocellular marrows (93% of all patients), in vast contrast to the typical proliferative subtypes of AML in the elderly. Most patients had a normal karyotype (75%) and acquired a second DDX41 variant (69%). A favorable overall survival (OS) was observed in comparison to that of common subtypes of AML with wild-type DDX41 in age-matched patients. Our study demonstrated that the frequent germline pathogenic DDX41 variants characterized a clinically distinct AML entity. Features characteristic of DDX41-mutated AML include male predominance, often lack of FH, indolent course, low proliferative potential, frequent somatic DDX41 variants, and a favorable OS.

An unusual case of cutaneous blastic plasmacytoid dendritic cell neoplasm with concomitant B-cell lymphoproliferative disorder.

Blastic plasmacytoid dendritic cell neoplasm is a rare hematologic malignancy characterized by aggressive clinical behavior and frequent cutaneous involvement. We describe a case of a 64-year-old man with a rapidly enlarging subcutaneous forearm mass. Histologic examination of the excisional biopsy specimen revealed a diffuse proliferation of atypical hematolymphoid cells in the dermis extending to the deep subcutaneous soft tissues. Occasional aggregates of small lymphocytes were noted to be distributed within the mass. The tumor cells expressed CD4, CD45, CD56, CD123, and terminal deoxynucleotidyl transferase (TdT) but not CD3, CD20, or CD34. A diagnosis of blastic plasmacytoid dendritic cell neoplasm was rendered. Chromosome analysis revealed a 45 X, -Y karyotype. In addition, flow cytometry identified a small population of monoclonal B cells. A staging bone marrow aspirate and biopsy was performed, which showed normal cytogenetics and no evidence of involvement by blastic plasmacytoid dendritic cell neoplasm. Flow cytometric evaluation of the bone marrow revealed a CD5-negative, CD10-negative monoclonal B-cell population consistent with a B-cell lymphoproliferative disorder. This is a very unusual example of cutaneous blastic plasmacytoid dendritic cell neoplasm with a novel cytogenetic finding and concomitant B-cell lymphoproliferative disorder. Although previously not reported, our case shows that blastic plasmacytoid dendritic cell neoplasm may be associated with lymphoid malignancy. The relationship between the 2 neoplasms, however, is unclear. A high degree of suspicion and bone marrow examination in patients with a new diagnosis of blastic plasmacytoid dendritic cell neoplasm is required to avoid this potential diagnostic pitfall.

Real-world diagnostic testing patterns for assessment of ring sideroblasts and SF3B1 mutations in patients with newly diagnosed lower-risk myelodysplastic syndromes.

INTRODUCTION: The presence of ring sideroblasts (RS) and mutation of the SF3B1 gene are diagnostic of lower-risk (LR) myelodysplastic syndromes (MDS) and are correlated with favorable outcomes. However, information on testing and reporting in community-based clinical settings is scarce. This study from the Connect® MDS/AML Disease Registry aimed to compare the frequency of RS and SF3B1 reporting for patients with LR-MDS, before and after publication of the 2016 World Health Organization (WHO) MDS classification criteria. METHODS: Ring sideroblasts assessment and molecular testing data were collected from patients with LR-MDS at enrollment in the Registry. Patients enrolled between December 2013 and the data cutoff of March 2020 were included in this analysis. RESULTS: Among 489 patients with LR-MDS, 434 (88.8%) underwent RS assessment; 190 were assessed prior to the 2016 WHO guidelines (Cohort A), and 244 after (Cohort B). In Cohort A, 87 (45.8%) patients had RS identified; 29 (33.3%) patients had RS < 15%, none of whom underwent molecular testing for SF3B1. In Cohort B, 96 (39.3%) patients had RS identified; 31 (32.3%) patients had < 15% RS, with 13 undergoing molecular testing of which 10 were assessed for SF3B1. CONCLUSIONS: In the Connect® MDS/AML Registry, only 32% of patients with <15% RS underwent SF3B1 testing after the publication of the WHO 2016 classification criteria. There was no change in RS assessment frequency before and after publication, despite the potential impact on diagnostic subtyping and therapy selection, suggesting an unmet need for education to increase testing rates for SF3B1 mutations.

Detection and Quantification of FLT3 Internal Tandem Duplication Mutations Do Not Vary Significantly Between Whole Blood and Blast-Enriched Samples.

OBJECTIVES: Many commonly used FLT3 mutational assay protocols require a tedious blast enrichment step. We investigated whether elimination of this step would still give equivalent results and compared the accuracy of variant allele fraction (VAF) between polymerase chain reaction/capillary electrophoresis (PCR/CE) vs next-generation sequencing (NGS) methods. METHODS: Total leukocyte vs blast-enriched whole-blood aliquots were tested for FLT3 internal tandem duplication (ITD) and tyrosine kinase domain mutations by PCR/CE. VAF of the ITD mutations was also compared with NGS VAF. RESULTS: Blast-enriched vs total leukocyte specimens showed 100% concordance in the 25 positive specimens. VAF was consistently lower by NGS, with poorer fidelity to PCR/CE VAF as the ITD size increased. CONCLUSIONS: Our study supports elimination of the blast enrichment step without compromising results or sensitivity. In addition, since NGS shows a loose correlation with PCR/CE quantitative results, NGS VAF should not be reported for FLT3 ITDs.