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.

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.

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.

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.

Molecular profiling reveals a hypoxia signature in breast implant-associated anaplastic large cell lymphoma.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a recently characterized T-cell malignancy that has raised significant patient safety concerns and led to worldwide impact on the implants used and clinical management of patients undergoing reconstructive or cosmetic breast surgery. Molecular signatures distinguishing BIA-ALCL from other ALCLs have not been fully elucidated and classification of BIA-ALCL as a WHO entity remains provisional. We performed RNA sequencing and gene set enrichment analysis comparing BIA-ALCLs to non-BIA-ALCLs and identified dramatic upregulation of hypoxia signaling genes including the hypoxia-associated biomarker CA9 (carbonic anyhydrase-9). Immunohistochemistry validated CA9 expression in all BIA-ALCLs, with only minimal expression in non-BIA-ALCLs. Growth induction in BIA-ALCL-derived cell lines cultured under hypoxic conditions was proportional to up-regulation of CA9 expression, and RNA sequencing demonstrated induction of the same gene signature observed in BIA-ALCL tissue samples compared to non-BIA-ALCLs. CA9 silencing blocked hypoxia-induced BIA-ALCL cell growth and cell cycle-associated gene expression, whereas CA9 overexpression in BIA-ALCL cells promoted growth in a xenograft mouse model. Furthermore, CA9 was secreted into BIA-ALCL cell line supernatants and was markedly elevated in human BIA-ALCL seroma samples. Finally, serum CA9 concentrations in mice bearing BIA-ALCL xenografts were significantly elevated compared to control serum. Together, these findings characterize BIA-ALCL as a hypoxia-associated neoplasm, likely attributable to the unique microenvironment in which it arises. These data support classification of BIA-ALCL as a distinct entity and uncover opportunities for investigating hypoxia-related proteins such as CA9 as novel biomarkers and therapeutic targets in this disease.

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.

Pembrolizumab in patients with CLL and Richter transformation or with relapsed CLL.

Chronic lymphocytic leukemia (CLL) patients progressed early on ibrutinib often develop Richter transformation (RT) with a short survival of about 4 months. Preclinical studies suggest that programmed death 1 (PD-1) pathway is critical to inhibit immune surveillance in CLL. This phase 2 study was designed to test the efficacy and safety of pembrolizumab, a humanized PD-1-blocking antibody, at a dose of 200 mg every 3 weeks in relapsed and transformed CLL. Twenty-five patients including 16 relapsed CLL and 9 RT (all proven diffuse large cell lymphoma) patients were enrolled, and 60% received prior ibrutinib. Objective responses were observed in 4 out of 9 RT patients (44%) and in 0 out of 16 CLL patients (0%). All responses were observed in RT patients who had progression after prior therapy with ibrutinib. After a median follow-up time of 11 months, the median overall survival in the RT cohort was 10.7 months, but was not reached in RT patients who progressed after prior ibrutinib. Treatment-related grade 3 or above adverse events were reported in 15 (60%) patients and were manageable. Analyses of pretreatment tumor specimens from available patients revealed increased expression of PD-ligand 1 (PD-L1) and a trend of increased expression in PD-1 in the tumor microenvironment in patients who had confirmed responses. Overall, pembrolizumab exhibited selective efficacy in CLL patients with RT. The results of this study are the first to demonstrate the benefit of PD-1 blockade in CLL patients with RT, and could change the landscape of therapy for RT patients if further validated. This trial was registered at www.clinicaltrials.gov as #NCT02332980.

Genotype-Phenotype Correlation of Hereditary Erythrocytosis Mutations, a single center experience.

Hereditary erythrocytosis is associated with high oxygen affinity hemoglobin variants (HOAs), 2,3-bisphosphoglycerate deficiency and abnormalities in EPOR and the oxygen-sensing pathway proteins PHD, HIF2α, and VHL. Our laboratory has 40 years of experience with hemoglobin disorder testing and we have characterized HOAs using varied protein and molecular techniques including functional assessment by p50 analysis. In addition, we have more recently commenced adding the assessment of clinically relevant regions of the VHL, BPGM, EPOR, EGLN1 (PHD2), and EPAS1 (HIF2A) genes in a more comprehensive hereditary erythrocytosis panel of tests. Review of our experience confirms a wide spectrum of alterations associated with erythrocytosis which we have correlated with phenotypic and clinical features. Through generic hemoglobinopathy testing we have identified 762 patients with 81 distinct HOA Hb variants (61 ß, 20 α), including 12 that were first identified by our laboratory. Of the 1192 cases received for an evaluation specific for hereditary erythrocytosis, approximately 12% had reportable alterations: 85 pathogenic/likely pathogenic mutations and 58 variants of unknown significance. Many have not been previously reported. Correlation with clinical and phenotypic data supports an algorithmic approach to guide economical evaluation; although, testing is expanded if the suspected causes are negative or of uncertain significance. Clinical features are similar and range from asymptomatic to recurrent headaches, fatigue, restless legs, chest pain, exertional dyspnea and thrombotic episodes. Many patients were chronically phlebotomized with reported relief of symptoms. This article is protected by copyright. All rights reserved.

MYD88 mutation status does not impact overall survival in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is an immunoglobulin M-associated lymphoma, with majority of cases demonstrating MYD88 locus alteration, most commonly, MYD88L265P . Owing to low prevalence of the wild-type (WT) MYD88 genotype in WM, clinically relevant data in this patient population are sparse, with one study showing nearly a 10-fold increased risk of mortality in this subgroup compared to patients with MYD88L265P mutation. We studied a large cohort of patients with MYD88L265P and MYD88WT WM, evaluated at Mayo Clinic, Rochester, between 1995 and 2016, to specifically assess the impact of these genotypes on clinical course. Of 557 patients, MYD88L265P mutation status, as determined by allele-specific polymerase chain reaction, was known in 219, and 174 (79%) of those exhibited MYD88L265P , 157 of 174 patients had active disease. Of 45 (21%) patients with MYD88WT genotype, 44 had active disease. The estimated median follow-up was 7.0 years; median overall survival was 10.2 years (95% CI: 8.4-16.5) for MYD88L265P versus 13.9 years (95% CI: 6.4-29.3) for the MYD88WT (P = 0.86). The time-to-next therapy from frontline treatment and the presenting features were similar in the two patient populations. For patients with smoldering WM at diagnosis, the median time-to-progression to active disease was 2.8 years (95% CI: 2.2-3.8) in the MYD88L265P cohort and 1.9 years (95% CI: 0.7-3.1) in the MYD88WT cohort (P = 0.21). The frequency of transformation to high-grade lymphoma, or the development of therapy-elated myelodysplastic syndrome was higher in the MYD88WT cohort (16% versus 4% in the MYD88L265P , P = 0.009). In conclusion, MYD88L265P mutation does not appear to be a determinant of outcome, and its presence may not be a disease-defining feature in WM. Our findings warrant external validation, preferably through prospective studies.

Clinical and laboratory characteristics in congenital ANKRD26 mutation-associated thrombocytopenia: A detailed phenotypic study of a family.

The clinical and laboratory characteristics of patients with non-syndromic, autosomal dominant thrombocytopenia secondary to germ line ANKRD26 mutations appear to be heterogeneous. Except for a targeted molecular genotyping approach, there is no distinct clinical or laboratory phenotype that has been specifically associated with this particular gene mutation. Such heterogeneity could be due to variations in mutation and genetic background in different families. To understand the phenotypic heterogeneity, we thoroughly studied one affected family using the International Society for Thrombosis and Haemostasis bleeding assessment tool and both clinically validated standard and esoteric platelet testing (electron microscopy (EM) and flow cytometry). We found that decreased platelet aggregation with arachidonic acid and epinephrine agonists was common in affected family members. EM studies demonstrated persistent borderline low mean dense granules per platelet, decreased alpha granules and an increased canalicular network pattern in all affected members. Since these characteristics are subtle or non-pathognomonic, molecular testing for ANKRD26 mutation remains the most reliable test to render a diagnosis and should be considered when evaluating a patient or family with congenital thrombocytopenia, particularly if there is a history of myeloid neoplasms.

PREVALENCE OF MYD88 L265P MUTATION IN HISTOLOGICALLY PROVEN, DIFFUSE LARGE B-CELL VITREORETINAL LYMPHOMA.

PURPOSE: Myeloid differentiation primary response gene 88 (MYD88) is a universal adaptor protein in the innate immune system. When associated with a proline for leucine substitution mutation at position 265 (L265P), the protein becomes constitutively activated, amplifying the intracellular pro-inflammatory signal. Recently, we reported two cases of vitreoretinal lymphoma (VRL) that were positive for the mutation. The purpose of this study was to determine prevalence of the MYD88 L265P mutation in a larger series of VRL. METHODS: Retrospective chart review of 25 patients with histologically confirmed VRL evaluated at Mayo Clinic, Rochester, between January 2000 and March 2015. Paraffin-embedded blocks from the vitreous were submitted for polymerase chain reaction testing of the L265P mutation. RESULTS: The mutation was positive in 82.4% of all VRL cases and 86.7% of primary VRL cases. The minimum necessary DNA concentration needed for the polymerase chain reaction assay was 4.93 ng/mL. CONCLUSION: MYD88 gene analysis is a helpful ancillary tool for diagnosing VRL. It often requires fewer cells than flow cytometry or cytology and may be especially useful in early cases where a sufficient number of cells may not be available.

MyD-88 L265P mutations are present in some cases of vitreoretinal lymphoma.

PURPOSE: To determine whether Myd88 L265P mutations occur in diffuse large B-cell vitreoretinal lymphomas. METHODS: The formalin-fixed paraffin-embedded cells from three patients with classic clinical findings of vitreoretinal lymphoma that also had histologic confirmation were evaluated using a validated amplification-refractory mutation system polymerase chain reaction to determine the presence of the mutation. RESULTS: The 74 ± 2 base pair product seen from the mutated Myd-88 protein was noted in 2 of the 3 cases. CONCLUSION: Myd-88 L265P constitutive activating mutations are present in at least some cases of the diffuse large B-cell lymphoma form of vitreoretinal lymphoma. Further studies on the incidence of this mutation in retinal lymphomas are warranted.

Genome-wide analysis reveals recurrent structural abnormalities of TP63 and other p53-related genes in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.

Chemoimmunotherapy for relapsed/refractory and progressive 17p13-deleted chronic lymphocytic leukemia (CLL) combining pentostatin, alemtuzumab, and low-dose rituximab is effective and tolerable and limits loss of CD20 expression by circulating CLL cells.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) patients with purine analog refractory disease or TP53 dysfunction still have limited treatment options and poor survival. Alemtuzumab-containing chemoimmunotherapy regimens can be effective but frequently cause serious infections. We report a Phase II trial testing the efficacy and tolerability of a short-duration regimen combining pentostatin, alemtuzumab, and low-dose high-frequency rituximab designed to decrease the risk of treatment-associated infections and to limit the loss of CD20 expression by CLL cells. The study enrolled 39 patients with progressive CLL that was either relapsed/refractory (n = 36) or previously untreated with 17p13 deletion (17p13-) (n = 3). Thirteen (33%) patients had both 17p13- and TP53 mutations predicted to be dysfunctional, and eight patients had purine analog refractory CLL without TP53 dysfunction. Twenty-six (67%) patients completed therapy, with only five (13%) patients having treatment-limiting toxicity and no treatment-related deaths. Twenty-two (56%) patients responded to treatment, with 11 (28%) complete responses (four with incomplete bone marrow recovery). Median progression-free survival was 7.2 months, time to next treatment was 9.1 months, and overall survival was 34.1 months. The majority of deaths (82%) were caused by progressive disease, including transformed diffuse large B-cell lymphoma (n = 6). Correlative studies showed that low-dose rituximab activates complement and natural killer cells without a profound and sustained decrease in expression of CD20 by circulating CLL cells. We conclude that pentostatin, alemtuzumab, and low-dose high-frequency rituximab is a tolerable and effective therapy for CLL and that low-dose rituximab therapy can activate innate immune cytotoxic mechanisms without substantially decreasing CD20 expression.

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.