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Introduction: Multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) can both be used to identify a neoplastic clonotype by targeting CDR3 and assessing rearrangements in IgH, IgK, IgL, TCR-β, and TCR-gamma loci. The clonotypic sequence can be robustly used to track minimal residual disease (MRD). The ability to track MRD by NGS in mixed phenotype acute leukemia (MPAL) is unknown and warrants investigation., Methods: Institutional Review Board (IRB) approval was obtained. Central Moffitt Cancer Center (MCC) database was searched to locate any patients with MPAL from over 600,000 entries. Patient charts were manually curated to identify those with clonoSEQ data, and clinical data was procured from the electronic medical record (EMR)., Results: Twenty-nine patients with MPAL were identified. Only 2 patients with clonoSEQ testing were found. Both demonstrated a B/myeloid phenotype, and both were bilineal. NGS (clonoSEQ) identified 4 dominant (IGH) (patient A; 8/2019) and 2 dominant sequences (patient B; 10/2019), respectively. In both patients, clonoSEQ testing successfully tracked minimal residual disease and mirrored clinical disease burden., Conclusions: This report is the first to confirm the utility of NGS-based MRD tracking in patients with MPAL and shows increased sensitivity of NGS over MRD flow cytometry., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction/Background The impact of biological sex on the clinical phenotype, genotype, and outcomes among patients with MDS is not well characterized. Materials and Methods We retrospectively reviewed the clinical and genomic data from male and female patients included in our institutional MDS database at Moffitt Cancer Center. Results Among 4580 patients with MDS, 2922 (66%) were men and 1658 (34%) were women. Women were younger (mean age 66.5 vs. 69 years for men, P < .001) at diagnosis. There were more Hispanic/black women than men (9% vs. 5%, P =<.001). Women had lower hemoglobin and higher platelet counts than men. More women had del 5q/monosomy 5 abnormalities compared to men (P =<.001). Therapy related MDS were more common in women than men (25% vs.17%, P=<.001). On assessment of molecular profile, SRSF2, U2AF1, ASXL1, and RUNX1 mutations were more frequent in men. The median overall survival (mOS) was 37.5 months (mo) for females compared to 35 monthsfor males, (P = .002). The mOS was significantly prolonged for women in lower-risk MDS, but not in higher-risk MDS. Women were more likely to respond to immunosuppression with ATG/CSA than men (38% vs. 19%, P= 0.04).Conclusion Ongoing research is needed for understanding the impact of sex on phenotype, genotype, and outcomes in patients diagnosed with MDS., (Copyright © 2023 Elsevier Inc. All rights reserved.)

EZH2 mutations in myeloid neoplasms are loss of function type, and have been linked to poor overall survival (OS) in patients with myelodysplastic syndrome (MDS). However, the specific determinants of outcomes in EZH2-mutant (mut) MDS are not well characterized. In this single-center retrospective study, clinical and genomic data were collected on 1774 patients with MDS treated at Moffitt Cancer Center. In our cohort, 83 (4.7%) patients had a pathogenic EZH2 mutation. Patients with EZH2mut MDS were older than EZH2-wild type (wt) group (median age- 72 vs. 69 years, p = 0.010). The most common co-occurring mutation in EZH2mut MDS was ASXL1, with a significantly higher frequency than EZH2wt (54% vs. 19%, p < 0.001). Patients with EZH2mut MDS had lower response rates to hypomethylating agents compared to EZH2wt MDS (26% vs. 39%; p = 0.050). Median OS of patients with EZH2mut MDS was 30.8 months, with a significantly worse OS than EZH2wt group (35.5 vs. 61.2 months, p = 0.003) in the lower-risk IPSS-R categories. Among patients with EZH2mut MDS, co-presence of ASXL1 or RUNX1 mutations was associated with inferior median OS compared to their wt counterparts (26.8 vs. 48.7 months, p = 0.031). Concurrent chromosome 7 abnormalities (12%) were also associated with significantly worse OS (median OS- 20.8 vs. 35.5 months, p = 0.002) in EZH2mut MDS. Future clinical trials should explore the potential role of novel targeted therapies in improving outcomes in patients with EZH2mut MDS., Competing Interests: Conflict of Interest: Hussaini Consultancy/Advisory Board/Speaking for Adaptive Biotechnologies, Amgen, Aptitude Health, Bluprint Oncology, Celgene, Decibio, Diaceutics, Guidepoint, Seattle Genetics, Stemline, Tegus, Janssen. Tinsley-Vance: Jazz: Consultancy, Speakers Bureau; Taiho: Consultancy; Abbvie: Honoraria; Astellas: Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Novartis: Consultancy; Fresenius Kabi: Consultancy. Kuykendall: BluePrint Medicines: Honoraria, Research Funding, Speakers Bureau; Celgene/BMS: Honoraria, Research Funding, Speakers Bureau; CTI Biopharma: Honoraria; Incyte: Consultancy; Novartis: Honoraria, Speakers Bureau; PharmaEssentia: Honoraria; Prelude: Research Funding; Abbvie: Honoraria; Protagonist: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sweet: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Lancet: AbbVie: Consultancy; Millennium Pharma/Takeda: Consultancy; Daiichi Sankyo: Consultancy; BerGenBio: Consultancy; ElevateBio Management: Consultancy; Astellas: Consultancy; Agios: Consultancy; Celgene/BMS: Consultancy; Jazz: Consultancy. Sallman: Takeda: Consultancy; Agios: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Intellia: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta: Consultancy. Padron: Blueprint: Honoraria; Taiho: Honoraria; Incyte: Research Funding; BMS: Research Funding; Stemline: Honoraria; Kura: Research Funding. Komrokji: Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; AbbVie: Consultancy; Acceleron: Consultancy; Jazz: Consultancy, Speakers Bureau; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Ball, Aguirre, Jain, Ali, Chan, and Padron: No conflict of interest to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Genomic landscape of extramedullary acute myeloid leukemia (EM-AML), including myeloid sarcoma (MS) and leukemia cutis (LC), is not well characterized. The potential utility of next-generation sequencing (NGS) using EM tissue is not established., Methods: In this multicenter retrospective study, clinical and NGS data were collected on patients with EM-AML. All statistical analyses were performed in SPSS Statistics (v 26)., Results: Our study included 58 patients with EM-AML. The median age at diagnosis was 62 years; 59% of patients had MS and 33% had LC. EM-AML was isolated (i.e., without blood or marrow involvement) in 31% and was first noted at relapse in 60% of patients. Median overall survival in our cohort was 18.2 months overall, with 19.1 months and 11.6 months in the newly diagnosed and the relapsed/refractory patients, respectively. At least one targetable or potentially targetable alteration was present in 52% of patients with EM-site NGS, with 26% IDH1, 21% NPM1, 11% IDH2, 6% FLT3, and 13% KMT2A-PTD. Mutations in IDH1 were significantly more prevalent on NGS from EM tissue than non-EM (blood or marrow) samples (26% vs. 3%; p = .030). Three of four patients treated with IDH inhibitors based on EM-site NGS experienced a complete response., Conclusions: Targetable mutations are frequent in EM-AML and EM-site NGS is warranted for selecting potential targeted therapies for patients with EM-AML., (© 2022 American Cancer Society.)

Context.—: Measurable (minimal) residual disease (MRD) is an independent prognostic factor for survival outcomes in patients with lymphoid and plasma cell malignancies and has been incorporated into consensus criteria regarding treatment response, strategy, and clinical trial endpoints. clonoSEQ (a next-generation sequencing [NGS]-MRD assay) uses multiplex polymerase chain reaction and NGS to identify clonotypic rearrangements at the immunoglobulin (Ig) H, IgK, IgL, T-cell receptor (TCR)-β, and TCR-γ loci, as well as translocated B-cell lymphoma 1/IgH and 2/IgH sequences for MRD assessment. Additionally, it can be used to confirm diagnoses of cutaneous T-cell lymphoma (CTCL)., Objective.—: To review the technical aspects of our experience using the clonoSEQ Assay in routine clinical practice., Design.—: In this single-center experience, 390 patients with lymphoid and plasma cell malignancies were assessed with the NGS-MRD Assay at a central laboratory., Results.—: Median time from arrival of the shipment to initiation of the assay (defined as captured in Adaptive's secure tracking system) was 2.1 hours. Overall, 317 patients had 1 or more samples submitted for sequence identification. Of these, 290 (91.5%) had trackable sequences identified. The median calibration rate of samples by malignancy (where n ≥ 10 samples, excluding CTCL samples) was 88.1%, across a variety of fresh and archived sample sources (177 of 201 samples). TCR-β and/or TCR-γ clonotypes were identified in 40 of 95 samples (42.1%) from 66 patients with suspected CTCL., Conclusions.—: This NGS-MRD Assay is a valuable and sensitive tool for monitoring MRD in patients with plasma cell and lymphoid malignancies and assisting in the diagnosis of CTCL., Competing Interests: Hussaini declares participating on advisory councils or committees for, and receiving honoraria from, Adaptive Biotechnologies Corp and consulting fees from Amgen. Srivastava and Lee are employees of, and hold stocks/shares with, Adaptive Biotechnologies Corp. Shah declares participating on advisory councils or committees for Adaptive Biotechnologies Corp. Pinilla-Ibarz declares receipt of honoraria from Adaptive Biotechnologies Corp. Shain declares receiving honoraria from Amgen, Inc, and consulting fees from Adaptive Biotechnologies Corp. Alsina has no relevant financial interest in the products or companies described in this article.

Objectives: The presence of targeted therapy, Enasidenib, for IDH2-mutated AML underscores the importance of understanding the clonal dynamics of IDH2 mutations, which has not been elucidated. In the largest study of IDH2 clonal dynamics, we detail the IDH2-evolutionary patterns and their clinical significance., Methods: We analyzed ~6000 patients with NGS results to identify 120 AML patients with IDH2 mutations and longitudinal NGS testing. IDH2 mutation status was recorded at diagnosis, remission, relapse, and persistent disease., Results: One hundred and five patients were IDH2-positive at the initial diagnosis, and 15 acquired the mutation later. Of those 15 patients, 7 patients gained the mutation during persistent disease, 6 during relapse, and 2 at remission. Twenty-one patients (18%) who were IDH2-positive in a prior test remained IDH2-positive in remission. Twenty-four patients with IDH2-positive AML were IDH2-positive at relapse. IDH2-positive at diagnosis had better survival than IDH2 mutation acquired later in disease (P = .024). Patients who were IDH2-negative in remission had significantly improved survival (P = .002). Also, loss-of-IDH2 mutation with persistent disease had better OS (P = .035)., Conclusions: There are 70% that clear IDH2 in disease remission. 12% gain IDH2 mutation later, usually in the setting of refractory/relapsed AML. These patients fared worse. Longitudinal IDH2 testing may be helpful in prognostic stratification., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The ASXL1 and SRSF2 mutations in AML are frequently found in patients with preexisting myeloid malignancies and are individually associated with poor outcomes. In this multi-institutional retrospective analysis, we assessed the genetic features and clinical outcomes of 43 patients with ASXL1 mut SRSF2 mut AML and compared outcomes to patients with either ASXL1 (n = 57) or SRSF2 (n = 70) mutations. Twenty-six (60%) had secondary-AML (s-AML). Variant allele fractions suggested that SRSF2 mutations preceded ASXL1 mutational events. Median overall survival (OS) was 7.0 months (95% CI:3.8,15.3) and was significantly longer in patients with de novo vs s-AML (15.3 vs 6.4 months, respectively; P = .04 on adjusted analysis). Compared to ASXL1 mut SRSF2 wt and ASXL1 wt SRSF2 mut , co-mutated patients had a 1.4 and 1.6 times increase in the probability of death, respectively (P = .049), with a trend towards inferior OS (median OS = 7.0 vs 11.5 vs 10.9 months, respectively; P = .10). Multivariable analysis suggests this difference in OS is attributable to the high proportion of s-AML patients in the co-mutated cohort (60% vs 32% and 23%, respectively). Although this study is limited by the retrospective data collection and the relatively small sample size, these data suggest that ASXL1 mut SRSF2 mut AML is a distinct subgroup of AML frequently associated with s-AML and differs from ASXL1 mut SRSF2 wt /ASXL1 wt SRSF2 mut with respect to etiology and leukemogenesis., (© 2021 Wiley Periodicals LLC.)

A subset of AML with myelodysplastic syndrome (MDS)-related changes (MRCs) occurs without a documented MDS phase. We studied genomic profile of 646 patients: 310 with MDS, 167 with AML without (w/o) MRC, 99 with primary (p) AML-MRC, and 70 with secondary (s) AML-MRC and sought to find differences in mutational patterns. Among the 32-myeloid associated genes studied, SF3B1 ( p  ≤ .001) was significantly mutated in higher proportion of patients with MDS, compared to other categories. NPM1 ( p  < .001), FLT3 ITD ( p  = .08), and NRAS ( p  = .02) mutations showed trend toward significance for AML w/o MRC, compared to other categories. In pAML-MRC, TP53 ( p  < .001) was significantly mutated in higher proportion of patients. Similarly, SETBP1 ( p  = .001), RUNX1 ( p  = .004), and SRSF2 ( p  = .04) mutations were more commonly seen in sAML-MRC. While these signatures may not be diagnostically discriminatory, they may help in disease categorization when other data are absent or in challenging cases.

Introduction: The role of single mutations has been extensively studied myelodysplastic syndromes (MDS), but the impact of genetic aberrations in the context of other mutations is less well understood. BCOR is an epigenetic transcriptional corepressor. In MDS, BCOR mutations are rare and certain mutations are associated with poor prognosis. Our aim was to investigate the role of concurrent mutations in epigenetic MDS-driver genes in BCOR -mutated MDS. We hypothesised that these would be redundant and would not contribute to worse prognosis., Methods: Internal Next Generation Sequencing (NGS) database with targeted genetic profiling of >4000 tumor cases was queried to locate cases of MDS with BCL6 Corepressor protein ( BCOR ) mutations only (pBCOR) and cBCOR (comutated epigenetic modulators: TET2 , ASXL1 , DNMT3A , EZH2 , IDH2 , IDH1 , BCORL1 , ATRX ). Overall survival was determined by chart review. Fischer's exact test and unpaired t-test was performed for statistical analysis., Results: 25 patients with cBCOR were detected. Only five MDS patients with pBCOR were found. The number of patients with comutations (cBCOR) in epigenetic modulators comprised TET2 (n=5), ASXL1 (n=9), DNMT3A (n=11), EZH2 (n=2), IDH2 (n=4), IDH1 (n=1), BCORL1 (n=3), ATRX (n=1). cBCOR overall survival was 23.8 months versus 11.8 months for pBCOR group., Conclusions: In this study, we confirm the rarity of BCOR mutations. Our results show that there is a trend towards poorer prognosis in patient with pBCOR versus cBCOR although statistical significance was not reached. This may be due to enrichment of poor cytogenetics in pBCOR or increased responsiveness to hypomethylating agents in cBCOR. Larger studies are needed to validate our data., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Although several technologies can be used to detect gene fusions, anchored multiplex PCR next-generation sequencing (AMP-NGS) offers the advantage of novel fusion detection and the ability to multiplex multitudinous genes. We applied AMP-NGS technology in the evaluation of a 56-year-old gentleman with myelodysplastic syndrome transformed acute myeloid leukemia (AML). Patient was initially diagnosed with low-risk myelodysplastic syndrome-refractory cytopenias and multilineage dysplasia (MDS-RCMD), progressed to AML after failing hypomethylating agent therapy. At progression patients had normal cytogenetics but NGS profiling showed ETV6 c.416&#95;417del CT frame shift and U2AF1 S34F mutations. Patient attains brief remission of 2 months after induction chemotherapy and then he was refractory to 2 salvage chemotherapy regimens. Reassessment after failing second salvage, identified t(12;17)(p13;p13)[20] by karyotype. It was postulated that the 12p13 locus might represent a new rearrangement of ETV6. AMP-NGS confirmed involvement of the ETV6 with discovery of a novel fusion partner, HIC1. The detection of the novel fusion partners was supported by the breakpoints originally observed by karyotype. This discovery of ETV6-HIC1 gene fusion by AMP-NGS technology provided new insight into a leukemogenic pathway in AML. Future use of this technology can serve as an adjunct tool in workup of patients with AML and can also help in formulating therapeutic strategies.

Background/aim: Acute myeloid leukemia (AML) represents a heterogeneous disease with varying morphologic, immunophenotypic, and genetic features, along with varying patient outcomes. The genomic tractability of AML makes it amenable for targeted next-generation sequencing (NGS) testing clinically., Materials and Methods: One hundred eights-seven unique patients with a diagnosis of acute myeloid leukemia between May 2011 and Oct 2014 and with mutational analysis by NGS were included in this study. The distribution of gene mutations was investigated in different subcategories of AML., Results: Most patients in this study (n=182) received Genoptix testing (either 5-gene panel or 21-gene panel). In 130/187 (70%) cases, there was an average of 2.3 mutations per case (range=0-7 mutations). We specifically mention mutations in 32 genes, their significance and co-occurrence as detected in different types of AML., Conclusion: The genetic heterogeneity of AML signifies the importance of taking a personalized-medicine approach to the management of patients with AML., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Aim: To report the first case of a Richter syndrome where small lymphocytic lymphoma (SLL) progressed to a CD3+ diffuse large B-cell lymphoma (DLBCL)., Methods: Macrodissection of small and large cell lymphomatous components was performed. This was followed by flow cytometric analysis along with molecular B-cell immunoglobulin (heavy and light chains) and T-cell receptor (γ and β chains) gene rearrangement studies to investigate a clonal relationship between the components., Results: The immunophenotypic profile was similar between small and large cell components of the lymphoma by flow cytometry. Furthermore, shared clonal peaks were observed between both components based on molecular B-cell and T-cell receptor gene rearrangement studies, confirming a clonal relationship., Conclusions: Chronic lymphocytic leukaemia/SLL may rarely undergo Richter transformation to a DLBCL demonstrating lineage infidelity. This is a potentially important diagnostic pitfall and such cases should not be confused with a de novo T-cell lymphoma., Competing Interests: Competing interests: None., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

CD4 expression is rare in diffuse large B-cell lymphoma (DLBCL), with 4 previously reported cases. Its significance is uncertain. We report five patients with CD4(+) DLBCL and one CD4(+) primary mediastinal large B-cell lymphoma. Cases were identified by searching the electronic database of the department; each was reviewed. Average age was 56 years. Neoplastic cells expressed CD20 (5/6 tested cases). BCL2/BCL6 expression were seen in 3/3 tested cases, suggesting a germinal center origin. Additionally, expression of T-cell antigens CD2 and CD5 was noted in 2/2 and CD7 in 1/1 tested case. CD3 was negative in all. Lymph nodes were commonly involved (67%). Patients received chemotherapy +/- radiation (6/6) and bone marrow transplant (2/6). Average survival was 44.2 mo. CD4 expression in DLBCL raises questions of lineage commitment. CD4(+) DLBCL is rare; care should be exercised not to diagnose these as T-cell lymphomas. A subset behaves aggressively., Competing Interests: Conflict-of-interest statement: The authors declare that they have no conflict of interest.