TP53 variant allele frequency and therapy-related setting independently predict survival in myelodysplastic syndromes with del(5q).

Among 210 patients with myelodysplastic syndromes (MDSs) with del(5q), molecular information was available at diagnosis or at least 3 months before leukaemic transformation in 146 cases. Multivariate analysis identified therapy-related setting (p = 0.02; HR 2.3) and TP53 variant allele frequency (VAF) ≥22% (p < 0.01; HR 2.8), but not SF3B1 mutation (p = 0.65), as independent risk factors for survival. Median survival was 11.7 versus 4 years (5/10-year survival 73%/52% vs. 42%/14%) in the absence (N = 112) versus presence (N = 34) of ≥1 risk factors; leukaemia-free survival was affected by TP53 VAF ≥22% (p < 0.01). Such information might inform treatment decision-making in MDS-del(5q) regarding allogeneic stem cell transplant.

VEXAS syndrome: Clinical, hematologic features and a practical approach to diagnosis and management.

VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a newly identified disease caused by somatic alterations in UBA1 which produce a recalcitrant inflammatory state along with hematologic disturbances. Patients with VEXAS can have a wide spectrum of clinical symptoms and providers should be familiar with the heterogeneity of associated clinical features. While hematologic parameters may be generally non-specific, peripheral blood features of macrocytosis, monocytopenia, and/or thrombocytopenia coupled with bone marrow vacuolization of erythroid or myeloid precursors should raise suspicion for this condition. Due to an increased mortality, prompt recognition and accurate diagnosis is paramount. Access to testing for confirmation of UBA1 variants is not yet universally available but clinicians should understand the current available options for genetic confirmation of this disease. Treatment options are limited due to lack of prospective clinical trials but cytokine directed therapies such as interleukin-6 inhibitors and JAK-STAT inhibitors as well as hypomethylating agents such as azacitidine have shown evidence of partial effect. Though cases are limited, allogeneic stem cell transplantation holds promise for durable response and potential cure. The intent of this review is to outline the pathophysiology of VEXAS syndrome and to provide a practical approach to diagnosis and treatment.

Granularity in disease classification impacts survival prediction in advanced systemic mastocytosis: A single institution study of 329 informative cases.

The World Health Organization (WHO) classification system categorizes advanced systemic mastocytosis (SM-Adv) into aggressive SM (ASM), mast cell leukemia (MCL), and SM with associated hematological neoplasm (SM-AHN). By contrast, the International Consensus Classification (ICC) requires "immature" MC cytomorphology for the diagnosis of MCL and limits SM-AHN to myeloid neoplasms (SM-AMN). The current study includes 329 patients with SM-Adv (median age 65 years, range 18-88; males 58%): WHO subcategories SM-AHN (N = 212; 64%), ASM (N = 99; 30%), and MCL (N = 18; 6%); ICC subcategories SM-AMN (N = 190; 64%), ASM (N = 99; 33%), and MCL (N = 9; 3%); WHO-defined MCL with "mature" MC cytomorphology and SM-AHN associated with lymphoid neoplasms were operationally labeled as "MCL-mature" (N = 9) and SM-ALN (N = 22), respectively, and distinguished from ICC-defined MCL and SM-AMN. Multivariable analysis that included the Mayo alliance risk factors for survival in SM (age >60 years, anemia, thrombocytopenia, increased alkaline phosphatase) revealed more accurate survival prediction with the ICC versus WHO classification order: (i) survival was significantly worse with MCL-immature versus MCL-mature (hazard ratio [HR] 15; p < .01), (ii) prognostic distinction between MCL and SM-AHN/AMN was confirmed in the context of ICC (HR 9.3; p < .01) but not WHO classification order (p = .99), (iii) survival was similar between MCL-mature and SM-AMN (p = .18), and (iv) SM-AMN (HR 1.7; p < .01) but not SM-ALN (p = .37) was prognostically distinct from ASM. The current study provides evidence for the independent prognostic contribution of both the ICC system for SM-Adv and the Mayo alliance risk factors for survival in SM.

Reappraisal of mast cell leukemia based on a single institution review of 16 cases: Mast cell morphology determines clinical outcome.

Cytologic abnormalities of atypical mast cells in mastocytosis. The mature mast cells have oval-shaped nuclei, cytoplasmic hypogranulation and spindle-shaped cytology. or well-differentiated displaying a round nucleus with condensed chromatin, and abundant dense cytoplasmic granulations. Immature mast cells include promastocytes and metachromatic blast-like forms.

Targeted testing of bone marrow specimens with cytoplasmic vacuolization to identify previously undiagnosed cases of VEXAS syndrome.

OBJECTIVE: To retrospectively identify patients with VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome) among male patients with bone marrow vacuolization using a clinically applicable, targeted-screening approach. METHODS: Bone marrow reports from 1 May 2014 through 18 February 2022 were reviewed for documentation of cytoplasmic vacuolization. Patients with acute leukaemia, lymphoma, metastatic solid tumour, amyloidosis or POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes) syndrome were excluded, as were those without clinical records available for direct chart review. Cases were rated for suspicion of VEXAS syndrome using a 5-point scale based on the presence of laboratory findings, clinical features and treatment response. Patients with available DNA material and moderate (three patients) or high (four to five patients) suspicion were tested for somatic UBA1 variants associated with VEXAS syndrome. RESULTS: A total of 315 reports from 292 unique patients included documentation of vacuolization. Following exclusion criteria, 64 patients underwent direct medical chart review to assess likelihood of VEXAS syndrome, for which 21 patients met moderate to high suspicion. Available DNA was present in eight patients, seven (87.5%) of whom had a pathogenic somatic UBA1 variant consistent with VEXAS syndrome. The distribution of cytoplasmic vacuolization in the bone marrow biopsy reports among patients with VEXAS syndrome were erythroid and myeloid precursors (6/7), erythroid precursors only (1/7) and myeloid precursors only (0/7). CONCLUSION: In this study, the utilization of a clinically applicable targeted-screening approach to test bone marrow specimens (with vacuolization) for the presence of previously undiagnosed VEXAS syndrome resulted in a positive detection rate of 87.5%.

Typical, atypical and cryptic t(15;17)(q24;q21) (PML::RARA) observed in acute promyelocytic leukemia: A retrospective review of 831 patients with concurrent chromosome and PML::RARA dual-color dual-fusion FISH studies.

The diagnosis of acute promyelocytic leukemia (APL) relies on the identification of PML::RARA fusion. While the majority of APL cases harbor a typical t(15;17)(q24;q21), atypical genetic mechanisms leading to the oncogenic PML::RARA fusion have been reported yet their frequency and scope remain poorly characterized. We assessed the genetic findings of 831 cases with APL investigated with concurrent chromosome banding analysis and dual-color dual-fusion fluorescence in situ hybridization (D-FISH) analysis at our institution over an 18.5-year timeframe. Seven hundred twenty-three (87%) cases had a typical balanced t(15;17) with both testing modalities. Atypical karyotypic results including complex translocations, unbalanced rearrangements and insertional events occurred in 50 (6%) cases, while 6 (0.7%) cases were cryptic by conventional chromosome studies despite PML::RARA fusion by D-FISH evaluation. Atypical FISH patterns were observed in 48 (6%) cases despite apparently balanced t(15;17) on chromosome banding analysis. Two hundred fifty (30%) cases displayed additional chromosome abnormalities of which trisomy/tetrasomy 8 (37%), del(7q)/add(7q) (12%), and del(9q) (7%) were most frequent. Complex and very complex karyotypes were observed in 81 (10%) and 34 (4%) cases, respectively. In addition, 4 (0.5%) cases presented as an apparently doubled, near-tetraploid stemline clone. This report provides the largest appraisal of cytogenetic findings in APL with conventional chromosome and PML::RARA D-FISH analysis. By characterizing the frequency and breadth of typical and atypical results through the lens of these cytogenetic testing modalities, this study serves as a pragmatic source of information for those involved in the investigation of APL in both the clinical and research laboratory settings.

Cladribine therapy for advanced and indolent systemic mastocytosis: Mayo Clinic experience in 42 consecutive cases.

We describe our single institution experience with cladribine therapy in 42 patients with systemic mastocytosis (SM): 22 advanced (adv-SM; median age 65 years, 68% males) and 20 indolent/smouldering SM (ISM/SSM; median age 56 years, 45% males); subcategories included eight aggressive, 13 associated with another haematological neoplasm, one mast cell leukaemia, 17 ISM and three SSM. Overall/major response rates were 77%/45% for adv-SM and 70%/60% for ISM/SSM, and median (range) duration of response 10 (4-75) and 46 (4-140) months respectively. A >50% reduction in bone marrow mast cell burden and serum tryptase level was documented in 63% and 67% of patients with adv-SM and 50% and 46% with ISM/SSM respectively. The presence of KIT proto-oncogene, receptor tyrosine kinase (KIT)D816V predicted response in adv-SM: 17 (90%) of 19 with and none of three without the mutation responded (P < 0·01). Treatment-emergent adverse events were mostly limited to transient cytopenias: Grade 3/4 neutropenia, thrombocytopenia, or lymphopenia occurred in 27%, 27% and 27% of patients with adv-SM, and 5%, 5% and 30% with ISM/SSM respectively. The present study provides practical information that might be considered when making treatment choices between cladribine and newer KIT-targeted therapies and identifies the absence of KITD816V as a potential marker of cladribine resistance in advanced SM; the latter observation needs confirmation in a larger study.

Midostaurin therapy for advanced systemic mastocytosis: Mayo Clinic experience in 33 consecutive cases.

We retrospectively examined our experience with midostaurin therapy in 33 consecutive patients (median age 68 years; 58% females) with advanced systemic mastocytosis (adv-SM): aggressive SM (ASM; n = 17), SM associated with another hematologic neoplasm (SM-AHN; n = 14) and mast cell leukemia (MCL; n = 2). KITD816V mutation was detected in 84% of the patients and C findings in 91%. Eleven (33%) patients were previously treated with other cytoreductive drugs, including cladribine (n = 4) and imatinib (n = 3). Median time from diagnosis to initiation of midostaurin therapy was 2.2 months (range 0.3-41). Using modified valent criteria, overall response was 42% (53% ASM, 29% SM-AHN, 50% MCL; p = .22), all classified as being major. Responses included ≥50% reduction in bone marrow mast cells in 40% and normalization of serum tryptase in 29%, of evaluated cases. After a median follow-up of 14.6 months from initiation of midostaurin therapy, 7 (21%) deaths, 1 (3%) leukemic progression, and 18 (55%) treatment discontinuations were documented; median duration of midostaurin treatment was 7.9 months (range 0.5-123) and response duration 21.5 months (range 2.9-123). Most frequent side effect was gastrointestinal (51%) while grade 3/4 neutropenia or thrombocytopenia occurred in 12% of patients. Response to treatment was not predicted by KIT mutation (p = .67) or exposure to prior cytoreductive therapy (p = .44). Median survival was longer in midostaurin responders but not significantly (median 26.5 vs. 16 months; p = .15). Findings from the current study are broadly consistent with previously published clinical trial observations.

Identification of a novel KMT2A/GIMAP8 gene fusion in a pediatric patient with acute undifferentiated leukemia.

Acute undifferentiated leukemia (AUL) is a very rare hematologic neoplasm that expresses no markers specific for either myeloid or lymphoid lineages. While commonly observed in several acute leukemias, KMT2A rearrangements in AUL have been rarely reported in the literature. We report the third case to our knowledge of AUL harboring a KMT2A rearrangement. Furthermore, the KMT2A/GIMAP8 gene fusion identified in this case represents a novel KMT2A rearrangement.

Chronic myeloid neoplasms harboring concomitant mutations in myeloproliferative neoplasm driver genes (JAK2/MPL/CALR) and SF3B1.

JAK2, CALR, and MPL are myeloproliferative neoplasm (MPN)-driver mutations, whereas SF3B1 is strongly associated with ring sideroblasts (RS) in myelodysplastic syndrome (MDS). Concomitant mutations of SF3B1 and MPN-driver mutations out of the context of MDS/MPN with RS and thrombocytosis (MDS/MPN-RS-T) are not well-studied. From the cases (<5% blasts) tested by NGS panels interrogating at least 42 myeloid neoplasm-related genes, we identified 18 MDS/MPN-RS-T, 42 MPN, 10 MDS, and 6 MDS/MPN-U cases with an SF3B1 and an MPN-driver mutation. Using a 10% VAF difference to define "SF3B1-dominant," "MPN-mutation dominant," and "no dominance," the majority of MDS/MPN-RS-T clustered in "SF3B1-dominant" and "no dominance" regions. Aside from parameters as thrombocytosis and ≥15% RS required for RS-T, MDS also differed in frequent neutropenia, multilineage dysplasia, and notably more cases with <10% VAF of MPN-driver mutations (60%, p = 0.0346); MPN differed in more frequent splenomegaly, myelofibrosis, and higher VAF of "MPN-driver mutations." "Gray zone" cases with features overlapping MDS/MPN-RS-T were observed in over one-thirds of non-RS-T cases. This study shows that concomitant SF3B1 and MPN-driver mutations can be observed in MDS, MPN, and MDS/MPN-U, each showing overlapping but also distinctively different clinicopathological features. Clonal hierarchy, cytogenetic abnormalities, and additional somatic mutations may in part contribute to different disease phenotypes, which may help in the classification of "gray zone" cases.

Hybridization capture-based next generation sequencing reliably detects FLT3 mutations and classifies FLT3-internal tandem duplication allelic ratio in acute myeloid leukemia: a comparative study to standard fragment analysis.

FLT3-internal tandem duplication occurs in 20-30% of acute myeloid leukemia and confers an adverse prognosis with its allelic ratio being a key risk stratifier. The US Food and Drug Administration recently approved FLT3 inhibitors midostaurin and gilteritinib in FLT3 mutation-positive acute myeloid leukemia. Historically, FLT3 was tested by fragment analysis, which has become the standard method endorsed by international guidelines. However, next generation sequencing is increasingly used at acute myeloid leukemia diagnosis given its ability to simultaneously evaluate multiple clinically informative markers. As FLT3-internal tandem duplication detection was known to be challenging by next generation sequencing and the results carry profound prognostic and therapeutic implications, it is important to thoroughly examine its performance in FLT3-internal tandem duplication detection and allelic ratio classification. In a comparative study with fragment analysis, we retrospectively reviewed our experience using a custom-designed, hybridization capture-based, targeted next generation sequencing panel. Among 7902 cases, FLT3-internal tandem duplication was detected in 335 with variable sizes (3-231 bp) and insertion sites. Fragment analysis was also performed in 402 cases, demonstrating 100% concordance in FLT3-internal tandem duplication detection. In 136 dual-tested, positive cases, 128/136 (94%) exhibited concordant high/low allelic ratio classifications. The remaining 6% showed borderline low allelic ratio by next generation sequencing. The two methods were concordant in FLT3-tyrosine kinase domain mutation detection at the hotspot D835/I836 targeted by fragment analysis. Furthermore, seven mutations which may benefit from FLT3 inhibitor therapy were detected by next generation sequencing, in regions not covered by fragment analysis. Our study demonstrates that using a hybridization capture-based chemistry and optimized bioinformatics pipeline, next generation sequencing can reliably detect FLT3-internal tandem duplication and classify its allelic ratio for acute myeloid leukemia risk stratification. Next generation sequencing also exhibits superior comprehensiveness in FLT3 mutation detection and may further improve personalized, targeted therapy in acute myeloid leukemia.

Correction to: Hybridization capture-based next-generation sequencing reliably detects FLT3 mutations and classifies FLT3-internal tandem duplication allelic ratio in acute myeloid leukemia: a comparative study to standard fragment analysis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Systematic use of fluorescence in-situ hybridisation and clinicopathological features in the screening of PDGFRB rearrangements of patients with myeloid/lymphoid neoplasms.

AIMS: Rearrangement of the platelet-derived growth factor receptor B (PDGFRB) gene defines a unique group of myeloid/lymphoid neoplasms with frequent eosinophilia and high sensitivity to tyrosine kinase inhibitors. This genetic abnormality is also rarely reported in Philadelphia-like B-cell acute lymphoblastic leukaemia/lymphoma (B-ALL). PDGFRB rearrangement was initially thought to only occur in cases with 5q31-33 rearrangement as determined with conventional cytogenetics; however, there are reported cases with cryptic rearrangements. We aim to develop a broader strategy for screening of PDGFRB rearrangements of patients with myeloid/lymphoid neoplasms. METHODS AND RESULTS: We performed fluorescence in-situ hybridisation (FISH) for PDGFRB rearrangement in 197 patients, including 70 with B-ALL, 10 with myeloid neoplasms with 5q31-33 rearrangements, and 117 with eosinophilia (≥0.5 × 109 /l in peripheral blood or ≥5% in bone marrow), and identified PDGFRB rearrangement in four of 197 (2.0%) cases. In an attempt to identify clinicopathological and genetic features that may have a stronger association with PDGFRB rearrangement, we analysed 13 patients with confirmed PDGFRB rearrangements, including 10 with myeloid neoplasms and three with B-ALL. Among the 10 patients with myeloid neoplasms, eosinophilia was present in eight, monocytosis in two, 5q31-33 rearrangement in seven, and abnormal bone marrow morphology in all. All patients with myeloid neoplasms showed an excellent response to imatinib, including a patient in blast crisis. The three B-ALL patients presented de novo, showed no eosinophilia, had a complex karyotype including 5q31-33 rearrangement, and had clinically aggressive courses with ultimate patient demise. CONCLUSIONS: These findings suggest that a higher yield for the identification of PDGFRB rearrangement may result from an index of suspicion in patients with eosinophilia, monocytosis, bone marrow features of a myeloid neoplasm, and 5q31-33 rearrangement, and patients with Philadelphia-like B-ALL.

IGH translocations in chronic lymphocytic leukemia: Clinicopathologic features and clinical outcomes.

The prevalence, clinicopathologic correlates, and outcomes of previously untreated chronic lymphocytic leukemia (CLL) patients with IGH-BCL2 and IGH-BCL3 translocations are not well known. Using the Mayo Clinic CLL database, we identified patients seen between March 1, 2002 and September 30, 2016 who had FISH testing performed within 3 years of CLL diagnosis. The prognostic profile, time to first therapy (TTT), and overall survival (OS) of patients with IGH-BCL2 and IGH-BCL3 translocation were compared to patients without these abnormalities (non-IGH group). Of 1684 patients who met the inclusion criteria, 38 (2.2%) had IGH-BCL2, and 16 (0.9%) had IGH-BCL3 translocation at diagnosis. Patients with IGH-BCL3 translocation were more likely to have high and very-high CLL-International Prognostic Index, compared to patients with IGH-BCL2 translocation and the non-IGH group. The 5-year probability of requiring therapy was significantly higher for IGH-BCL3 compared to IGH-BCL2 and non-IGH groups (84% vs 33% vs 29%, respectively, P < 0.0001). The 5-year OS was significantly shorter for IGH-BCL3 compared to IGH-BCL2 and non-IGH groups (45% vs 89% vs 86%, respectively, P < 0.0001). On multivariable analyses, IGH-BCL3 translocation was associated with a shorter TTT (hazard ratio [HR] = 2.7; P = 0.005) and shorter OS (HR = 5.5; P < 0.0001); IGH-BCL2 translocation did not impact TTT and OS. In conclusion, approximately 3% of all newly diagnosed CLL patients have either an IGH-BCL2 or IGH-BCL3 translocation. Patients with IGH-BCL3 translocations have a distinct prognostic profile and outcome. These results support the inclusion of an IGH probe during the routine evaluation of FISH abnormalities in newly diagnosed CLL.

De novo pure erythroid leukemia: refining the clinicopathologic and cytogenetic characteristics of a rare entity.

Per the revised fourth edition World Health Organization classification of acute myeloid leukemia, pure erythroid leukemia is now the sole type of acute erythroid leukemia. The diagnosis of this rare entity is often challenging and the cytologic overlap with non-neoplastic (eg, megaloblastic anemia) and neoplastic entities (eg, other types of acute leukemia and non-hematopoietic malignancies) warrants a significant degree of clinical, laboratory, immunophenotypic, and genetic investigation. Given the limited number of reports of this rare and diagnostically challenging entity, we report detailed clinicopathologic characteristics from 15 patients, the largest series thus far, of primary de novo pure erythroid leukemia to provide further diagnostic insights into this entity and reveal strategies for making the diagnosis. We found that de novo pure erythroid leukemia is a disease of adults (median age 68 years), exhibits a striking male predominance, is universally associated with an abnormal karyotype and has an exceedingly poor overall median survival of 1.4 months. Given the general inability of immunophenotypic markers to discriminate neoplastic from non-neoplastic erythroid proliferations, key features identified in this study to help establish the diagnosis of pure erythroid leukemia and exclude mimickers include circulating pronormoblasts, clear-cut dysplasia in erythroid, granulocytic, and/or megakaryocytic lineage, utilization of a broad immunophenotypic panel, TP53 immunohistochemical positivity, and identification of a complex, often highly complex, karyotype. Given the gravity of a diagnosis of de novo pure erythroid leukemia, it should be rendered with utmost confidence.

Cytogenetic abnormalities in systemic mastocytosis: WHO subcategory-specific incidence and prognostic impact among 348 informative cases.

The World Health Organization (WHO) system lists five morphological categories of systemic mastocytosis (SM): indolent (ISM), smoldering, SM with an associated hematological neoplasm (SM-AHN), aggressive (ASM) and mast cell leukemia (MCL). Recent studies have highlighted the prognostic importance of mutations in SM, including ASXL1, RUNX1, and SRSF2. In contrast, information on incidence of cytogenetic abnormalities in SM and their prognostic relevance, especially in the context of mutations, is limited. In the current study, we retrospectively reviewed the cytogenetic findings in 348 consecutive cases of SM (median age 59 years; 53% males); 41% constituted ISM, 45% SM-AHN, 14% ASM and two cases of MCL. Karyotype was abnormal in 53 (15%) cases with incidences of 6% for ISM, 26% for SM-AHN and 8% for ASM (P < .001); among SM-AHN cases, abnormal karyotype incidences were 0% for SM-AHN-lymphoid and 28% for SM-AHN-myeloid (P < .001). Clinical correlative studies disclosed significant associations between abnormal karyotype and male sex (P = .002), age > 60 years (P = .04), thrombocytopenia (P < .001) and anemia (P < .001), but not with the presence of adverse mutations (P = .19). In univariate analysis, abnormal karyotype was associated with inferior survival (HR 3.0, 95% CI 2.0-4.3), specifically confirmed for ASM (HR 4.9, 95% CI 1.1-16.1) and SM-AHN (HR 1.8, 95% CI 1.2-2.7). Sample size adequacy allowed additional multivariable analysis in SM-AHN-myeloid, which disclosed independent prognostic contribution from adverse mutations (P = .003), anemia (P = .003) and thrombocytopenia (P = .001), but not from abnormal karyotype (P = .31). Our observations suggest that mutations are prognostically more relevant than karyotype in SM.