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Christoph Plass, Christopher Oakes and colleagues study genome-wide DNA methylation dynamics during B cell maturation and the pathogenic role of transcription factor dysregulation in chronic lymphocytic leukemia (CLL). By comparing normal and malignant B cells, they find that tumors derive from a continuum of maturation states, which correlate with different clinical outcomes. Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program.

Chronic lymphocytic leukemia (CLL) is associated with perturbed immune function and increased risk for second primary malignancies (SPM). Ibrutinib and acalabrutinib (BTKi) are effective therapies for CLL resulting in partial restoration of immune function. The incidence of and risk factors for SPM in CLL patients receiving BTKi are not yet characterized. We retrospectively determined the incidence of SPM in CLL patients treated with ibrutinib or acalabrutinib at our institution between 2009 and 2017, assessed for association between baseline characteristics and SPM incidence, and compared the observed to expected cancer incidence among age, sex, and year matched controls without CLL. After a median of 44 months follow-up, 64/691 patients (9%) were diagnosed with SPM (excluding non-melanoma skin cancer [NMSC]). The 3-year cumulative incidence rate was 16% for NMSC and 7% for other SPM. On multivariable analysis, smoking was associated with increased SPM risk (HR 2.8 [95% CI: 1.6–4.8]) and higher baseline CD8 count was associated with lower SPM risk (HR 0.9 for 2-fold increase [95% CI: 0.8–0.9]). The observed over expected rate of SPM was 2.2 [95% CI: 1.7–2.9]. CLL patients treated with BTKi remain at increased risk for SPM, and secondary cancer detection is an important consideration in this population.

This study outlines the development of a microRNA signature in patients with cytogenetically normal acute myeloid leukemia (AML) with high-risk molecular features. This type of AML constitutes approximately 65% of cases of cytogenetically normal AML and one third of all AML cases involving patients under the age of 60 years. The microRNA signature correlated not only with the clinical outcome but also with the expression of genes encoding proteins of the innate immune system. This study outlines the development of a microRNA signature in patients with cytogenetically normal AML with high-risk molecular features. The microRNA signature correlated not only with the clinical outcome but also with the expression of genes encoding proteins of the innate immune system. In almost half of patients with acute myeloid leukemia (AML), no cytogenetic abnormality is detectable in the leukemic cells. Such patients are in an intermediate-risk prognostic category, 1 but among them are subgroups of patients who have molecular markers associated with either a favorable prognosis or an unfavorable prognosis. 2 Gene-expression profiling can also identify subgroups of patients who have cytogenetically normal AML with different outcomes. 3 – 5 Patients with internal tandem duplication in the fms-related tyrosine kinase 3 gene ( FLT3 -ITD) and those without FLT3 -ITD but with the wild-type nucleophosmin ( NPM1 ) gene are in a high-risk group, whereas . . .

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia and dysregulation of the T-cell leukemia/lymphoma 1 ( TCL1 ) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease based on transgenic mouse studies. To determine a role of microRNAs on the pathogenesis of the aggressive form of CLL we studied regulation of TCL1 expression in CLL by microRNAs. We identified miR-3676 as a regulator of TCL1 expression. We demonstrated that miR-3676 targets three consecutive 28-bp repeats within 3′UTR of TCL1 and showed that miR-3676 is a powerful inhibitor of TCL1 . We further showed that miR-3676 expression is significantly down-regulated in four groups of CLL carrying the 11q deletions, 13q deletions, 17p deletions, or a normal karyotype compared with normal CD19 ⁺ cord blood and peripheral blood B cells. In addition, the sequencing of 539 CLL samples revealed five germ-line mutations in six samples (1%) in miR-3676 . Two of these mutations were loss-of-function mutations. Because miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 ( Tp53 ), and is codeleted with Tp53 , we propose that loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression. Significance B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia. We previously found that dysregulation of the T-cell leukemia/lymphoma 1 ( TCL1 ) oncogene is a critical contributing event in the pathogenesis of this disease. In this study we investigated molecular causes of TCL1 overexpression in CLL. We identified miR-3676 as a powerful regulator of TCL1 expression. We found that miR-3676 is down-regulated on all groups of CLLs and mutated in 1% of CLLs. Interestingly, miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 ( Tp53 ), and is codeleted with Tp53 in 17p-deleted CLL. Loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression. Thus, this study uncovers a major mechanism in the pathogenesis of CLL.

Studies of chronic lymphocytic leukemia (CLL) have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL) generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient's CLL (CD5 positive with trisomy 12 and 19). A companion cell line was also generated from the same patient (OSU-NB). This cell line lacked typical CLL characteristics, and is likely derived from the patient's normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.

Significance IGHV mutation status is a well established prognostic factor in chronic lymphocytic leukemia, and also provides crucial insights into tumor cell biology and function. Currently, determination of IGHV transcript sequence, from which mutation status is calculated, requires a specialized laboratory procedure. RNA sequencing is a method that provides high resolution, high dynamic range transcriptome data that can be used for differential expression, isoform discovery, and variant determination. In this paper, we demonstrate that unselected next-generation RNA sequencing can accurately determine the IGH@ sequence, including the complete sequence of the complementarity-determining region 3 (CDR3), and mutation status of CLL cells, potentially replacing the current method which is a specialized, single-purpose Sanger-sequencing based test. Immunoglobulins (Ig) are produced by B lymphocytes as secreted antibodies or as part of the B-cell receptor. There is tremendous diversity of potential Ig transcripts (>1 × 10 ¹²) as a result of hundreds of germ-line gene segments, random nucleotide incorporation during joining of gene segments into a complete transcript, and the process of somatic hypermutation at individual nucleotides. This recombination and mutation process takes place in the maturing B cell and is responsible for the diversity of potential epitope recognition. Cancers arising from mature B cells are characterized by clonal production of Ig heavy ( IGH@ ) and light chain transcripts, although whether the sequence has undergone somatic hypermutation is dependent on the maturation stage at which the neoplastic clone arose. Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and arises from a mature B cell with either mutated or unmutated IGH@ transcripts, the latter having worse prognosis and the assessment of which is routinely performed in the clinic. Currently, IGHV mutation status is assessed by Sanger sequencing and comparing the transcript to known germ-line genes. In this paper, we demonstrate that complete IGH@ V-D-J sequences can be computed from unselected RNA-seq reads with results equal or superior to the clinical procedure: in the only discordant case, the clinical transcript was out-of-frame. Therefore, a single RNA-seq assay can simultaneously yield gene expression profile, SNP and mutation information, as well as IGHV mutation status, and may one day be performed as a general test to capture multidimensional clinically relevant data in CLL.

Clinical bleeding events are reported here from 773 patients with B‐cell malignancies receiving pirtobrutinib monotherapy from the phase 1/2 BRUIN study (ClinicalTrials.gov identifier: NCT03740529), either in the presence or absence of antithrombotic therapy (antithrombotic exposed [AT‐E], n = 216; antithrombotic nonexposed [AT‐NE], n = 557). Among the AT‐E cohort, 51.9% received platelet aggregation inhibitors, 36.6% received direct factor Xa inhibitors, 18.5% received heparins, 5.6% received salicylic acid for indications other than platelet aggregation inhibition, and 2.3% received thrombolytics. Warfarin was not permitted. Any‐grade bleeding/bruising events occurred in 97 patients (44.9%; 95% confidence interval [CI], 38.3–51.5) in the AT‐E cohort and 181 patients (32.5%; 95% CI, 28.6–36.4) in the AT‐NE cohort. Most bleeding/bruising events in both cohorts began within the first 6 months of treatment (AT‐E: 65.4%; AT‐NE: 72.5%). Contusion was the most common bleeding/bruising event in both cohorts (AT‐E: 22.7%; AT‐NE: 18.1%). Grade ≥3 bleeding/bruising events were reported in six patients (2.8%) in the AT‐E cohort and 11 patients (2.0%) in the AT‐NE cohort. Bleeding/bruising events requiring or prolonging hospitalization were reported in 2.3% and 1.6% of patients in the AT‐E and AT‐NE cohorts, respectively. No bleeding/bruising events led to pirtobrutinib dose reduction or permanent discontinuation in the AT‐E cohort, and one patient (0.2%) in the AT‐NE cohort experienced an event requiring dose reduction. These data support the safety of pirtobrutinib in patients requiring antithrombotic therapies.

Background Quantitative methods of Fluorodeoxyglucose Positron Emission Tomography (FDG‐PET) interpretation, including the percent change in FDG uptake from baseline (ΔSUV), are under investigation in lymphoma to overcome challenges associated with visual scoring systems (VSS) such as the Deauville 5‐point scale (5‐PS). Methods In CALGB 50303, patients with DLBCL received frontline R‐CHOP or DA‐EPOCH‐R, and although there were no significant associations between interim PET responses assessed centrally after cycle 2 (iPET) using 5‐PS with progression‐free survival (PFS) or overall survival (OS), there were significant associations between central determinations of iPET ∆SUV with PFS/OS. In this patient cohort, we retrospectively compared local vs central iPET readings and evaluated associations between local imaging data and survival outcomes. Results Agreement between local and central review was moderate (kappa = 0.53) for VSS and high (kappa = 0.81) for ∆SUV categories (<66% vs. ≥66%). ∆SUV ≥66% at iPET was significantly associated with PFS (p = 0.03) and OS (p = 0.002), but VSS was not. Associations with PFS/OS when applying local review vs central review were comparable. Conclusions These data suggest that local PET interpretation for response determination may be acceptable in clinical trials. Our findings also highlight limitations of VSS and call for incorporation of more objective measures of response assessment in clinical trials. In this retrospective analysis of CALGB 50303 study, Torka et al. found that associations with PFS and OS when applying local review versus central review of interim PET (iPET) were comparable? SUV = 66% at iPET was associated with PFS and OS, but visual scoring systems (VSS) were not, highlighting the limitations of VSS.

In patients previously treated with covalent, irreversible BTK inhibitors, pirtobrutinib (a noncovalent, reversible BTK inhibitor) induced responses in 73%, with a median progression-free survival of nearly 20 months.

In some patients with CLL, resistance to the BTK inhibitor ibrutinib develops. Two classes of resistance mutations have been found: the more common involves alteration of the drug-binding site to make binding reversible; the less common activates a downstream kinase that effectively bypasses BTK. The development of B-cell–receptor antagonists has been a therapeutic advance in chronic lymphocytic leukemia (CLL). Although B-cell–receptor ligation in normal cells induces proliferation, apoptosis, or anergy, 1 pathway dysregulation in CLL results in the propagation of proliferative and prosurvival signals. 2 , 3 Several agents targeting the B-cell–receptor pathway are in development, including the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. Although BTK is not recurrently mutated in CLL, 4 , 5 it is up-regulated at the transcript level and is constitutively active. 6 , 7 Ibrutinib irreversibly binds BTK at the C481 residue, rendering it kinase-inactive, inducing modest CLL-cell apoptosis, and abolishing proliferation and B-cell–receptor signaling in . . .