METTL3-Mediated m6A Modification Controls Splicing Factor Abundance and Contributes to Aggressive CLL.

RNA splicing dysregulation underlies the onset and progression of cancers. In chronic lymphocytic leukemia (CLL), spliceosome mutations leading to aberrant splicing occur in ∼20% of patients. However, the mechanism for splicing defects in spliceosome-unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis, we discover that proteins involved in RNA splicing are posttranscriptionally upregulated in CLL cells, resulting in splicing dysregulation. The abundance of splicing complexes is an independent risk factor for poor prognosis. Moreover, increased splicing factor expression is highly correlated with the abundance of METTL3, an RNA methyltransferase that deposits N6-methyladenosine (m6A) on mRNA. METTL3 is essential for cell growth in vitro and in vivo and controls splicing factor protein expression in a methyltransferase-dependent manner through m6A modification-mediated ribosome recycling and decoding. Our results uncover METTL3-mediated m6A modification as a novel regulatory axis in driving splicing dysregulation and contributing to aggressive CLL. SIGNIFICANCE: METTL3 controls widespread splicing factor abundance via translational control of m6A-modified mRNA, contributes to RNA splicing dysregulation and disease progression in CLL, and serves as a potential therapeutic target in aggressive CLL. See related commentary by Janin and Esteller, p. 176. This article is highlighted in the In This Issue feature, p. 171.

Harnessing the Power of Induced Pluripotent Stem Cells and Gene Editing Technology: Therapeutic Implications in Hematological Malignancies.

In vitro modeling of hematological malignancies not only provides insights into the influence of genetic aberrations on cellular and molecular mechanisms involved in disease progression but also aids development and evaluation of therapeutic agents. Owing to their self-renewal and differentiation capacity, induced pluripotent stem cells (iPSCs) have emerged as a potential source of short in supply disease-specific human cells of the hematopoietic lineage. Patient-derived iPSCs can recapitulate the disease severity and spectrum of prognosis dictated by the genetic variation among patients and can be used for drug screening and studying clonal evolution. However, this approach lacks the ability to model the early phases of the disease leading to cancer. The advent of genetic editing technology has promoted the generation of precise isogenic iPSC disease models to address questions regarding the underlying genetic mechanism of disease initiation and progression. In this review, we discuss the use of iPSC disease modeling in hematological diseases, where there is lack of patient sample availability and/or difficulty of engraftment to generate animal models. Furthermore, we describe the power of combining iPSC and precise gene editing to elucidate the underlying mechanism of initiation and progression of various hematological malignancies. Finally, we discuss the power of iPSC disease modeling in developing and testing novel therapies in a high throughput setting.

Advances in Diagnosis and Risk Stratification of Acute Myeloid Leukemia and Myelodysplastic Syndromes.

Advances in high-throughput DNA sequencing technology in the past decade have made a tremendous impact on basic science and clinical practice. Methods using the latest next generation sequencing technology can sequence an entire human genome within a few hours. Diagnosis and prognostication of hematologic neoplasms have moved from traditional histology and immunophenotyping to integration of cytogenetic and genomic alterations. Using illustrative cases, this chapter provides an overview of the utility of using genomic data for prognostication as well as treatment decision-making for patients with bone marrow neoplasms.

Genetics and Diagnostic Approach to Lymphoblastic Leukemia/Lymphoma.

Our understanding of the genetics and biology of lymphoblastic leukemia/lymphoma (acute lymphoblastic leukemia, ALL) has advanced rapidly in the past decade with advances in sequencing and other molecular techniques. Besides recurrent chromosomal abnormalities detected by karyotyping or fluorescence in situ hybridization, these leukemias/lymphomas are characterized by a variety of mutations, gene rearrangements as well as copy number alterations. This is particularly true in the case of Philadelphia-like (Ph-like) ALL, a major subset which has the same gene expression signature as Philadelphia chromosome-positive ALL but lacks BCR-ABL1 translocation. Ph-like ALL is associated with a worse prognosis and hence its detection is critical. However, techniques to detect this entity are complex and are not widely available. This chapter discusses various subsets of ALL and describes our approach to the accurate classification and prognostication of these cases.

Co-stimulatory and co-inhibitory immune markers in solid tumors with MET alterations.

The implication of MET alterations in solid tumors and the immune microenvironment remains elusive. Formalin-fixed, paraffin-embedded samples of 21 patients with solid tumors harboring MET alterations were used for immunohistochemical staining. Extracted RNA was analyzed with the NanoString nCounter human PanCancer immune profiling panel (NanoString Technologies, Inc., WA, USA). Patients were diagnosed with lung (n = 10), breast (n = 5), genitourinary (n = 3) or colorectal cancer (n = 3). Eleven had a MET missense mutation, four had an exon 14 splice site mutation and six had MET amplification. CD6, CCL19, CD40LG, XCR1, MAGEA1, ATM and CCL19 genes were significantly differentially expressed in MET-altered cancers. MET alterations may have a role in various solid tumors as potential therapeutic targets and combination therapy candidates with immune checkpoint inhibitors.

Allogeneic Hematopoietic Cell Transplantation Outcomes in Patients Carrying Isocitrate Dehydrogenase Mutations.

BACKGROUND: Mutations in isocitrate dehydrogenase (IDH)1/2 genes result in nicotinamide adenine dinucleotide phosphate-dependent reduction of α-ketoglutarate and formation of 2-hydroxyglutarate, which blocks normal cellular differentiation and promotes leukemogenesis. Nearly 20% of acute myeloid leukemia (AML) patients carry IDH1/2 mutations. Although multiple investigators have described the prognostic implications of IDH mutations in AML patients receiving chemotherapy, the effect of these mutations on outcomes after allogeneic (allo) hematopoietic cell transplantation (HCT) is unknown. PATIENTS AND METHODS: We report on the clinical outcome of a cohort of AML patients, who were tested for IDH mutations and underwent alloHCT at City of Hope (2015-2017). Of a total of 317 screened patients, 99 (31%) underwent alloHCT, of whom 23 carried and 76 did not carry IDH mutations (control). RESULTS: No statistical significance was detected in patient's overall survival (P = .84). With a median follow-up of 7.8 months, 1-year relapse rate of 29% and 13% was seen in the IDH-mutated and control group, respectively (P = .033). IDH1/2 mutation status remained significantly associated with relapse (hazard ratio, 2.8; P = .046) after inclusion of pre-HCT disease status in a multivariable model. CONCLUSION: Our results, despite low patient numbers, indicate that IDH mutations are associated with higher relapse rate after alloHCT. Further prospective studies on post transplantation IDH inhibition is required to improve outcomes in AML patients who carry IDH mutations.

B Cell Lymphoma Immunotherapy Using TLR9-Targeted Oligonucleotide STAT3 Inhibitors.

Growing evidence links the aggressiveness of non-Hodgkin's lymphoma, especially the activated B cell-like type diffuse large B cell lymphomas (ABC-DLBCLs) to Toll-like receptor 9 (TLR9)/MyD88 and STAT3 transcription factor signaling. Here, we describe a dual-function molecule consisting of a clinically relevant TLR9 agonist (CpG7909) and a STAT3 inhibitor in the form of a high-affinity decoy oligodeoxynucleotide (dODN). The CpG-STAT3dODN blocked STAT3 DNA binding and activity, thus reducing expression of downstream target genes, such as MYC and BCL2L1, in human and mouse lymphoma cells. We further demonstrated that injections (i.v.) of CpG-STAT3dODN inhibited growth of human OCI-Ly3 lymphoma in immunodeficient mice. Moreover, systemic CpG-STAT3dODN administration induced complete regression of the syngeneic A20 lymphoma, resulting in long-term survival of immunocompetent mice. Both TLR9 stimulation and concurrent STAT3 inhibition were critical for immune-mediated therapeutic effects, since neither CpG7909 alone nor CpG7909 co-injected with unconjugated STAT3dODN extended mouse survival. The CpG-STAT3dODN induced expression of genes critical to antigen-processing/presentation and Th1 cell activation while suppressing survival signaling. These effects resulted in the generation of lymphoma cell-specific CD8/CD4-dependent T cell immunity protecting mice from tumor rechallenge. Our results suggest that CpG-STAT3dODN as a systemic/local monotherapy or in combination with PD1 blockade can provide an opportunity for treating patients with B cell NHL.

Complete regression of cutaneous metastases with systemic immune response in a patient with triple negative breast cancer receiving p53MVA vaccine with pembrolizumab.

A heavily pretreated patient with triple negative breast cancer distinguished by cutaneous metastases received p53MVA vaccine in combination with pembrolizumab. Her cutaneous metastases regressed and after 2 cycles of therapy, a skin biopsy showed a complete pathological response. Systemic response was confirmed with restaging CT and bone scans. Activation of p53-specific T cell responses and elevation of multiple immune response genes in peripheral blood correlated with the rapid clinical response which lasted for 6 months after the initiation of combined therapy.

The State of the Art in Colorectal Cancer Molecular Biomarker Testing.

The number of molecular biomarkers to inform treatment decisions in patients with metastatic colorectal cancer (mCRC) continues to expand and with it the methodologies that can be employed to evaluate these biomarkers. Beyond standard diagnostic and prognostic biomarkers, such as those used for Lynch syndrome, mutations in KRAS exon 2 are well established as predictive for lack of response to the antiepidermal growth factor receptor therapies panitumumab and cetuximab. Recent studies have extended these findings by demonstrating that mutations in KRAS exons 3 and 4 and in NRAS exons 2, 3, and 4 (with all KRAS and NRAS mutations collectively referred to as RAS) are also predictive for treatment outcomes among patients with mCRC receiving panitumumab and cetuximab in combination with chemotherapy or as monotherapy. Consequently, evaluation of these additional loci has been incorporated into current clinical guidelines, and pathologists will need to develop testing procedures and algorithms to reliably and rapidly evaluate RAS status. With the increased number of mutations that must be examined to evaluate the status of RAS and other emerging biomarkers, next-generation sequencing technologies are likely to become increasingly important in mCRC testing. This review describes new considerations for pathologists that have arisen as a consequence of the incorporation of additional biomarker testing into clinical practice for mCRC.

Follicular lymphoma-like B cells of uncertain significance (in situ follicular lymphoma) may infrequently progress, but precedes follicular lymphoma, is associated with other overt lymphomas and mimics follicular lymphoma in flow cytometric studies.

In situ follicular lymphoma, more recently known as follicular lymphoma-like B cells of uncertain/undetermined significance is well accepted. However, the morphological criteria have evolved since it was first described and data are limited and conflicting regarding its clinical implications and whether the extent of involvement predicts an association with overt lymphoma. It is also unknown how often it will be identified by flow cytometric studies and how often it precedes overt follicular lymphomas. A multiparameter study of 31 biopsies with follicular lymphoma-like B cells of uncertain significance and 4 'benign' lymph node biopsies that preceded an overt follicular lymphoma was, therefore, performed. Fifty-two percent of biopsies with follicular lymphoma-like B cells were associated with a prior or concurrent lymphoma but only 6% subsequently developed lymphoma (median follow up 26 months). Neither the number, proportion or density of BCL2(+) germinal centers were associated with overt follicular lymphoma/diffuse large B-cell lymphoma. Flow cytometric studies identified follicular lymphoma-like B cells in 8 of 15 evaluable cases. The proportion but not the absolute number of BCL2(+) germinal centers was associated with the likelihood of positive flow cytometric studies (P<0.01). All 4 'benign' biopsies that preceded an overt follicular lymphoma demonstrated follicular lymphoma-like B cells. Thus, although few patients with follicular lymphoma-like B cells of uncertain significance progress within the follow-up period, it at least precedes many follicular lymphomas. The extent of involvement does not predict the occurrence of prior or concurrent lymphomas. Flow cytometric studies demonstrating follicular lymphoma-like B cells must not be over-interpreted as they may only reflect follicular lymphoma-like B cells.

Double-hit B-cell lymphomas with BCL6 and MYC translocations are aggressive, frequently extranodal lymphomas distinct from BCL2 double-hit B-cell lymphomas.

Double-hit (DH) lymphomas with MYC and either BCL2 (DH-BCL2/MYC) or BCL6 (DH-BCL6/MYC) rearrangements are considered very aggressive, many of which are now included in the category B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) (DLBCL/BL). However, data describing the DH cases are largely based on DH-BCL2/MYC cases. To better characterize DH-BCL6/MYC cases, the clinical, morphologic, phenotypic, and cytogenetic features of 6 cases from University of Pittsburgh Medical Center and 17 cases from the Mitelman database were reviewed. In the University of Pittsburgh Medical Center cases, the median age was 83 years (range, 51 to 89 y) with 5/6 DLBCL/BL cases and 1 large B-cell lymphoma, not otherwise specified. Five of 6 had a germinal center phenotype, 1/6 was BCL2(+), and the median Ki-67 score was 98% (35% to 100%). The Mitelman DH-BCL6/MYC cases included 13 aggressive B-cell lymphomas (diagnosed as DLBCL-5, BL-5, BL-like lymphomas-2, and primary effusion lymphoma-1) and 4 other lymphoid/plasmacytic neoplasms. The median cytogenetic complexity score was 2.5 (range, 0 to 14) in 14 evaluable mature aggressive lymphomas with an immunoglobulin gene partner for MYC in 9/14 and for BCL6 in 7/14 cases. Ten of 13 cases involved extranodal extramedullary sites at presentation, and the median survival for the 10 patients with large cell neoplasms or BL and with available follow-up data was 9 months. Thus, DH-BCL6/MYC lymphomas are aggressive, frequently involve extranodal sites, and are often DLBCL/BL with a germinal center phenotype. Unlike DH-BCL2/MYC lymphomas, however, they are more likely to be CD10(-) but IRF4/MUM-1(+) (P=0.03) and, more like BL, only infrequently express BCL2 (P<0.001), and are cytogenetically less complex (P<0.04).

Clonal T-cell receptor ß-chain gene rearrangements in differential diagnosis of lymphomatoid papulosis from skin metastasis of nodal anaplastic large-cell lymphoma.

BACKGROUND: In patients with a history of nodal anaplastic large-cell lymphoma (ALCL), differentiation of type C lymphomatoid papulosis from cutaneous involvement of systemic ALCL may be challenging because the 2 entities may exhibit identical histologic features. Although metastatic ALCL generally carries the same clone as the primary lymphoma, expression of a distinct clone likely represents a distinct process. OBSERVATIONS: A 54-year-old white man had a history of anaplastic lymphoma kinase 1-negative ALCL in the right inguinal lymph node 6 years ago. A complete response was achieved after 6 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine [Oncovin], and prednisone administered in 21-day cycles) and radiation therapy. After 3½ years, the patient observed waxing and waning papules and nodules. Examination of the biopsy specimen revealed a dense CD30(+) lymphocytic infiltrate; no evidence of systemic malignancy was evident on positron emission tomography. Although clinically the presentation was consistent with lymphomatoid papulosis, metastatic ALCL had to be excluded. Polymerase chain reaction analysis with T-cell receptor γ-chain gene rearrangement (TCR-γR) was performed on the original lymph node and new skin lesions. Results of the TCR-γR analysis were positive for clonality in both lesions. However, separate clonal processes were identified. The identification of distinct clones supported the clinical impression of lymphomatoid papulosis. CONCLUSION: Polymerase chain reaction analysis of TCR-γR is a useful method for distinguishing different clonal processes and is recommended when differentiation of primary and secondary lymphoproliferative disorders is required.