Detection of Hybrid Fusion Transcripts, Aberrant Transcript Expression, and Specific Single Nucleotide Variants in Acute Leukemia and Myeloid Disorders with Recurrent Gene Rearrangements.

INTRODUCTION: A variety of gene rearrangements and molecular alterations are key drivers in the pathobiology of acute leukemia and myeloid disorders; current classification systems increasingly incorporate these findings in diagnostic algorithms. Therefore, clinical laboratories require versatile tools, which can detect an increasing number and variety of molecular and cytogenetic alterations of clinical significance. METHODS: We validated an RNA-based next-generation sequencing (NGS) assay that enables the detection of: (i) numerous hybrid fusion transcripts (including rare/novel gene partners), (ii) aberrantly expressed EVI1 (MECOM) and IKZF1 (Del exons 4-7) transcripts, and (iii) hotspot variants in KIT, ABL1, NPM1 (relevant in the context of gene rearrangement status). RESULTS: For hybrid fusion transcripts, the assay showed 98-100% concordance for known positive and negative samples, with an analytical sensitivity (i.e., limit of detection) of approximately 0.8% cells. Samples with underlying EVI1 (MECOM) translocations demonstrated increased EVI1 (MECOM) expression. Aberrant IKZF1 (Del exons 4-7) transcripts detectable with the assay were also present on orthogonal reverse transcription PCR. Specific hotspot mutations in KIT, ABL1, and NPM1 detected with the assay showed 100% concordance with orthogonal testing. Lastly, several illustrative samples are included to highlight the assay's clinically relevant contributions to patient workup. CONCLUSION: Through its ability to simultaneously detect various gene rearrangements, aberrantly expressed transcripts, and hotspot mutations, this RNA-based NGS assay is a valuable tool for clinical laboratories to supplement other molecular and cytogenetic methods used in the diagnostic workup and in clinical research for patients with acute leukemia and myeloid disorders.

Current Landscape of Ancillary Diagnostic Testing in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is a heterogeneous disease with variable patient outcomes. A multidisciplinary technical evaluation, including flow cytometry, immunohistochemistry, molecular and cytogenetic analyses, can comprehensively characterize a patient's leukemia at diagnosis, identify important prognostic biomarkers, and track measurable residual disease; all of which can impact patient management. This review highlights the key concepts, clinical significance, and main biomarkers detectable with each of these technical approaches; the contents are a helpful resource for medical practitioners involved in the workup and management of patients with CLL.

Molecular profiling of the stroke-induced alterations in the cerebral microvasculature reveals promising therapeutic candidates.

Stroke-induced cerebral microvascular dysfunction contributes to aggravation of neuronal injury and compromises the efficacy of current reperfusion therapies. Understanding the molecular alterations in cerebral microvessels in stroke will provide original opportunities for scientific investigation of novel therapeutic strategies. Toward this goal, using a recently optimized method which minimizes cell activation and preserves endothelial cell interactions and RNA integrity, we conducted a genome-wide transcriptomic analysis of cerebral microvessels in a mouse model of stroke and compared these transcriptomic alterations with the ones observed in human, nonfatal, brain stroke lesions. Results from these unbiased comparative analyses have revealed the common alterations in mouse stroke microvessels and human stroke lesions and identified shared molecular features associated with vascular disease (e.g., Serpine1/Plasminogen Activator Inhibitor-1, Hemoxygenase-1), endothelial activation (e.g., Angiopoietin-2), and alterations in sphingolipid metabolism and signaling (e.g., Sphigosine-1-Phosphate Receptor 2). Sphingolipid profiling of mouse cerebral microvessels validated the transcript data and revealed the enrichment of sphingomyelin and sphingoid species in the cerebral microvasculature compared to brain and the stroke-induced increase in ceramide species. In summary, our study has identified novel molecular alterations in several microvessel-enriched, translationally relevant, and druggable targets, which are potent modulators of endothelial function. Our comparative analyses have revealed the presence of molecular features associated with cerebral microvascular dysfunction in human chronic stroke lesions. The results shared here provide a detailed resource for therapeutic discovery of candidates for neurovascular protection in stroke and potentially, other pathologies exhibiting cerebral microvascular dysfunction.

Acute hemolytic anemia after hematopoietic stem cell transplantation: An unusual invader.

69-year-old man with a history of diffuse large B-cell lymphoma (DLBCL) presented with severe acute hemolytic anemia 27 months after an autologous hematopoietic stem cell transplantation. Bone marrow aspirate revealed intracellular micro-organisms (arrows) located within the cytoplasm of red blood cells confirming the diagnosis of severe babesiosis.

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.

A FOXO1-dependent transcription network is a targetable vulnerability of mantle cell lymphomas.

Targeting lineage-defined transcriptional dependencies has emerged as an effective therapeutic strategy in cancer treatment. Through screening for molecular vulnerabilities of mantle cell lymphoma (MCL), we identified a set of transcription factors (TFs) including FOXO1, EBF1, PAX5, and IRF4 that are essential for MCL propagation. Integrated chromatin immunoprecipitation and sequencing (ChIP-Seq) with transcriptional network reconstruction analysis revealed FOXO1 as a master regulator that acts upstream in the regulatory TF hierarchy. FOXO1 is both necessary and sufficient to drive MCL lineage commitment through supporting the lineage-specific transcription programs. We further show that FOXO1, but not its close paralog FOXO3, can reprogram myeloid leukemia cells and induce B-lineage gene expression. Finally, we demonstrate that cpd10, a small molecule identified from an enriched FOXO1 inhibitor library, induces a robust cytotoxic response in MCL cells in vitro and suppresses MCL progression in vivo. Our findings establish FOXO1 inhibition as a therapeutic strategy targeting lineage-driven transcriptional addiction in MCL.

Comparison of Multiple Clinical Testing Modalities for Assessment of NPM1-Mutant AML.

BACKGROUND: NPM1 mutation status can influence prognosis and management in AML. Accordingly, clinical testing (i.e., RT-PCR, NGS and IHC) for mutant NPM1 is increasing in order to detect residual disease in AML, alongside flow cytometry (FC). However, the relationship of the results from RT-PCR to traditional NGS, IHC and FC is not widely known among many practitioners. Herein, we aim to: i) describe the performance of RT-PCR compared to traditional NGS and IHC for the detection of mutant NPM1 in clinical practice, and also compare it to FC, and ii) provide our observations regarding the advantages and disadvantages of each approach in order to inform future clinical testing algorithms. METHODS: Peripheral blood and bone marrow samples collected for clinical testing at variable time points during patient management were tested by quantitative, real-time, RT-PCR and results were compared to findings from a Myeloid NGS panel, mutant NPM1 IHC and FC. RESULTS: RT-PCR showed superior sensitivity compared to NGS, IHC and FC with the main challenge of NGS, IHC and FC being the ability to identify a low disease burden (<0.5% NCN by RT-PCR). Nevertheless, the positive predictive value of NGS, IHC and FC were each ≥ 80% indicating that positive results by those assays are typically associated with RT-PCR positivity. IHC, unlike bulk methods (RT-PCR, NGS and FC), is able provide information regarding cellular/architectural context of disease in biopsies. FC did not identify any NPM1-mutated residual disease not already detected by RT-PCR, NGS or IHC. CONCLUSION: Overall, our findings demonstrate that RT-PCR shows superior sensitivity compared to a traditional Myeloid NGS, suggesting the need for "deep-sequencing" NGS panels for NGS-based monitoring of residual disease in NPM1-mutant AML. IHC provides complementary cytomorphologic information to RT-PCR. Lastly, FC may not be necessary in the setting of post-therapy follow up for NPM1-mutated AML. Together, these findings can help inform future clinical testing algorithms.

Clinical Laboratory Validation and Implementation of Quantitative, Real-Time PCR-based Detection of NPM1 Type A Mutation.

BACKGROUND: NPM1 mutations have prognostic significance in acute myeloid leukemia (AML) and monitoring mutant NPM1 levels during and after therapy has been described to predict relapse and survival. Despite the published significance of this molecular biomarker, routine monitoring for mutant NPM1 levels has not been widely adopted in academic clinical laboratories. Therefore, our objective was to validate a quantitative, reverse transcription-PCR assay for the detection of NPM1 Type A mutant transcripts for use in the clinical laboratory. METHODS: A quantitative, real-time, reverse-transcription PCR-based method for the detection of NPM1 Type A mutant transcripts was validated for use in routine clinical practice. Results from this assay were compared to results from orthogonal methods, including next generation sequencing and digital droplet PCR. RESULTS: This real-time, reverse-transcription PCR-based method is sensitive (limit of detection: 0.0150% NCN and reproducible (≤ 0.5 log10-fold variation). We summarize the rigorous validation results and share observations that will help other clinical laboratories that may wish to implement this testing. We show the superior sensitivity of this assay compared to other assays (e.g., 45 gene Myeloid Next Generation Sequencing panel) and present a representative case which highlights the assay's utility in the pathologic assessment of cases with borderline morphologic or flow cytometric findings. CONCLUSIONS: As molecular testing for residual disease in AML continues to expand, this sensitive and reproducible method will be an appropriate testing option for the detection of NPM1 Type A mutant transcripts in clinical practice.

NPM1 Biology in Myeloid Neoplasia.

PURPOSE OF REVIEW: Nucleophosmin (NPM1) mutations are encountered in myeloid neoplasia and are present in ~ 30% of de novo acute myeloid leukemia cases. This review summarizes features of mutant NPM1-related disease, with a particular emphasis on recent discoveries relevant to disease monitoring, prognostication, and therapeutic intervention. RECENT FINDINGS: Recent studies have shown that HOX/MEIS gene overexpression is central to the survival of NPM1-mutated cells. Two distinct classes of small molecule drugs, BH3 mimetics and menin-MLL interaction inhibitors, have demonstrated exquisite leukemic cell toxicity in preclinical AML models associated with HOX/MEIS overexpression, and the former of these has shown efficacy in older treatment-naïve NPM1-mutated AML patients. The results of ongoing clinical trials further investigating these compounds will be of particular importance and may alter the clinical management of patients with NPM1-mutated myeloid neoplasms. Significant scientific advancements over the last decade, including improved sequencing and disease monitoring techniques, have fostered a much deeper understanding of mutant NPM1 disease biology, prognostication, and opportunities for therapeutic intervention. These discoveries have led to the development of clinical assays that permit the detection and monitoring of mutant NPM1 and have paved the way for future investigation of targeted therapeutics using emerging cutting-edge techniques.

Evaluation of S1PR1, pSTAT3, S1PR2, FOXP1 Expression in Aggressive, Mature B Cell Lymphomas.

BACKGROUND: Aggressive, mature B-cell lymphomas include Burkitt Lymphoma (BL), High Grade B Cell Lymphomas (HGBL) (eg, Double-Hit B cell lymphomas (HGBL-DH: HGBL with MYC and BCL2 and/or BCL6 translocations)), HGBL, Not Otherwise Specified (HGBL, NOS) and Diffuse Large B Cell Lymphoma (DLBCL). Overlapping morphologic and immunohistochemical features of these lymphomas pose diagnostic challenges in some cases, and better understanding of potential diagnostic biomarkers and possible therapeutic targets is needed. Sphingosine 1 Phosphate Receptors (S1PR1-5) are G-protein coupled receptors that bind S1P and influence migration and survival in multiple cell types, including lymphocytes. S1PRs are emerging as biomarkers in B cell biology and interaction between S1PR pathways and STAT3 or FOXP1 has been reported in DLBCL. AIM AND METHODS: Our aim was to extend the understanding of S1PR1, STAT3 and S1PR2, FOXP1 expression beyond DLBCL, into additional aggressive, mature B cell lymphomas using immunohistochemical expression analysis of human tissue samples. RESULTS: S1PR1 and S1PR2 showed different expression patterns in mantle zones and follicle centers in reactive lymphoid tissue. BL showed a unique expression pattern compared to HGBL and DLBCL. Additionally, S1PR1 and S1PR2 expression were typically mutually exclusive and were expressed in a low proportion of cases (frequently HGBL involving extranodal sites). FOXP1 was expressed in a high proportion of various case types and pSTAT3 was detected in a significant proportion of HGBL and DLBCL. CONCLUSIONS: These findings provide further evidence that S1PR1, pSTAT3, S1PR2 and FOXP1 play a role in a subset of aggressive, mature B cell lymphomas.

Expedited Analysis and Reporting of Multiple Mutations that Modify Medical Management of Myeloid Malignancies: It's About (Turnaround) Time!

This commentary highlights the article by Patel et al that reports a novel custom next-generation sequencing platform for fast detection of select genes in hematologic malignancies.

A B Cell Regulome Links Notch to Downstream Oncogenic Pathways in Small B Cell Lymphomas.

Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.

Molecular Pathology: Predictive, Prognostic, and Diagnostic Markers in Lymphoid Neoplasms.

Lymphoid neoplasms show great diversity in morphology, immunophenotypic profile, and postulated cells of origin, which also reflects the variety of genetic alterations within this group of tumors. This review discusses many of the currently known genetic alterations in selected mature B-cell and T-cell lymphoid neoplasms, and their significance as diagnostic, prognostic, and therapeutic markers. Given the rapidly increasing number of genetic alterations that have been described in this group of tumors, and that the clinical significance of many is still being studied, this is not an entirely exhaustive review of all of the genetic alterations that have been reported.

Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies.

Targeted next-generation sequencing panels to identify genetic alterations in cancers are increasingly becoming an integral part of clinical practice. We report here the design, validation, and implementation of a comprehensive 95-gene next-generation sequencing panel targeted for hematologic malignancies that we named rapid heme panel. Rapid heme panel is amplicon based and covers hotspot regions of oncogenes and most of the coding regions of tumor suppressor genes. It is composed of 1330 amplicons and covers 175 kb of genomic sequence in total. Rapid heme panel's average coverage is 1500× with <5% of the amplicons with <50× coverage, and it reproducibly detects single nucleotide variants and small insertions/deletions at allele frequencies of ≥5%. Comparison with a capture-based next-generation sequencing assay showed that there is >95% concordance among a wide array of variants across a range of allele frequencies. Read count analyses that used rapid heme panel showed high concordance with karyotypic results when tumor content was >30%. The average turnaround time was 7 days over a 6-month span with an average volume of ≥40 specimens per week and a low sample fail rate (<1%), demonstrating its suitability for clinical application.

An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma.

Translocation events are frequent in cancer and may create chimeric fusions or 'regulatory rearrangements' that drive oncogene overexpression. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in alternate ACC lineages.

MYC Immunohistochemistry to Identify MYC-Driven B-Cell Lymphomas in Clinical Practice.

OBJECTIVES: Immunohistochemistry with anti-MYC antibody (MYC IHC) detects MYC protein in fixed samples of aggressive B-cell lymphomas and, according to the number of positive staining tumor nuclei, facilitates tumor subclassification, predicts underlying MYC rearrangements, and stratifies patient outcome. We aimed to determine the performance of MYC IHC in clinical practice. METHODS: We reviewed MYC IHC performed on control specimens and 256 aggressive B-cell lymphomas and compared clinically reported IHC scores with experts' review. RESULTS: Control tissues showed less than 5% variation in daily IHC staining. Reported and expert IHC scores were well correlated (r = 0.86) with an SD of 14.2%. Reported IHC scores 30% or less and 70% or more were accurate (94.5%) compared with experts in categorizing tumors as "MYC IHC-Low" and "MYC IHC-High," respectively, but scores 40% to 60% were not (60.3%). The mean IHC score among lymphomas with MYC rearrangements was 80%, but with a large range of scores (20%-100%). There was no statistically significant association between IHC score and MYC copy number. CONCLUSIONS: Under optimal conditions, clinically reported MYC IHC scores are concordant with expert scores within 15%. MYC IHC does not capture all B-cell lymphomas with MYC rearrangements, however. MYC IHC and MYC fluorescence in situ hybridization are both recommended to identify MYC-driven B-cell lymphomas.

Critical role of sphingosine-1-phosphate receptor-2 in the disruption of cerebrovascular integrity in experimental stroke.

The use and effectiveness of current stroke reperfusion therapies are limited by the complications of reperfusion injury, which include increased cerebrovascular permeability and haemorrhagic transformation. Sphingosine-1-phosphate (S1P) is emerging as a potent modulator of vascular integrity via its receptors (S1PR). By using genetic approaches and a S1PR2 antagonist (JTE013), here we show that S1PR2 plays a critical role in the induction of cerebrovascular permeability, development of intracerebral haemorrhage and neurovascular injury in experimental stroke. In addition, inhibition of S1PR2 results in decreased matrix metalloproteinase (MMP)-9 activity in vivo and lower gelatinase activity in cerebral microvessels. S1PR2 immunopositivity is detected only in the ischemic microvessels of wild-type mice and in the cerebrovascular endothelium of human brain autopsy samples. In vitro, S1PR2 potently regulates the responses of the brain endothelium to ischaemic and inflammatory injury. Therapeutic targeting of this novel pathway could have important translational relevance to stroke patients.

Myeloid neoplasm demonstrating a STAT5B-RARA rearrangement and genetic alterations associated with all-trans retinoic acid resistance identified by a custom next-generation sequencing assay.

We describe the case of a patient presenting with several weeks of symptoms related to pancytopenia associated with a maturation arrest at the late promyelocyte/early myelocyte stage of granulocyte differentiation. A diagnosis of acute promyelocytic leukemia was considered, but the morphologic features were atypical for this entity and conventional tests for the presence of a PML-RARA fusion gene were negative. Additional analysis using a custom next-generation sequencing assay revealed a rearrangement producing a STAT5B-RARA fusion gene, which was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and supplementary cytogenetic studies, allowing the diagnosis of a morphologically atypical form of acute promyelocytic leukemia to be made. Analysis of the sequencing data permitted characterization of both chromosomal breakpoints and revealed two additional alterations, a small deletion in RARA exon 9 and a RARA R276W substitution, that have been linked to resistance to all-trans retinoic acid. This case highlights how next-generation sequencing can augment currently standard testing to establish diagnoses in difficult cases, and in doing so help guide selection of therapy.

Molecular classification of MYC-driven B-cell lymphomas by targeted gene expression profiling of fixed biopsy specimens.

Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are aggressive tumors of mature B cells that are distinguished by a combination of histomorphological, phenotypic, and genetic features. A subset of B-cell lymphomas, however, has one or more characteristics that overlap BL and DLBCL, and are categorized as B-cell lymphoma unclassifiable, with features intermediate between BL and DLBCL (BCL-U). Molecular analyses support the concept that there is a biological continuum between BL and DLBCL that includes variable activity of MYC, an oncoprotein once thought to be only associated with BL, but now recognized as a major predictor of survival among patients with DLBCL treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We tested whether a targeted expression profiling panel could be used to categorize tumors as BL and DLBCL, resolve the molecular heterogeneity of BCL-U, and capture MYC activity using RNA from formalin-fixed, paraffin-embedded biopsy specimens. A diagnostic molecular classifier accurately predicted pathological diagnoses of BL and DLBCL, and provided more objective subclassification for a subset of BCL-U and genetic double-hit lymphomas as molecular BL or DLBCL. A molecular classifier of MYC activity correlated with MYC IHC and stratified patients with primary DLBCL treated with R-CHOP into high- and low-risk groups. These results establish a framework for classifying and stratifying MYC-driven, aggressive, B-cell lymphomas on the basis of quantitative molecular profiling that is applicable to fixed biopsy specimens.