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Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell–like subtype (ABC) than germinal center B cell–like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL.Implications:The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Supplementary Table S1 including analyzed RNA-seq and ChIP-seq data

Supplementary Methods, Supplementary Figures and Figure Legends. Figure S1. EGR1 is a downstream target of BCR and JAK1 signaling (related to Figure 1). Figure S2. Sensitivity of MCL cell lines to EGR1 shRNAs and statistical analysis (related to Figure 2). Figure S3. EGR1 motif analysis and gene set enrichment analysis of RNA-seq data (related to Figure 3). Figure S4. Enrichment of E2F pathway genes in ABC DLBCL after EGR1 knockdown (related to Figure 4). Figure S5. EGR1 regulates expression of MYC and MYC targets and OCI-Ly10 xenografts (related to Figure 4). Figure S6. Enrichment of EGR1 on EGR1 own promoter and EP300 regulatory (5'UTR and enhancer) regions (related to Figure 4). Figure S7. Inhibition of EGR1 transcription and tumor growth by mithramycin (related to Figure 4). Figure S8. Synergism of mithramycin and lenalidomide in ABC DLBCL (related to Figure 6). Figure S9. Expression of MYC and MYC target genes is higher in ABC DLBCL than GCB DLBCL patient samples (related to the Discussion). Figure S10. No correlation between EGR1 and MYC expression in DLBCL cell lines and patient samples (related to the Discussion).

PDF file - 384K, Detection of multiple HPV subtypes by degenerate PCR of genomic DNA (A) or by HPV-16 specific Southern blot (B).

XLSX file - 37K, Primer sequences used.

Purpose: To develop a clinically relevant model system to study head and neck squamous cell carcinoma (HNSCC), we have established and characterized a direct-from-patient tumorgraft model of human papillomavirus (HPV)–positive and HPV-negative cancers.Experimental Design: Patients with newly diagnosed or recurrent HNSCC were consented for donation of tumor specimens. Surgically obtained tissue was implanted subcutaneously into immunodeficient mice. During subsequent passages, both formalin-fixed/paraffin-embedded as well as flash-frozen tissues were harvested. Tumors were analyzed for a variety of relevant tumor markers. Tumor growth rates and response to radiation, cisplatin, or cetuximab were assessed and early passage cell strains were developed for rapid testing of drug sensitivity.Results: Tumorgrafts have been established in 22 of 26 patients to date. Significant diversity in tumorgraft tumor differentiation was observed with good agreement in degree of differentiation between patient tumor and tumorgraft (Kappa 0.72). Six tumorgrafts were HPV-positive on the basis of p16 staining. A strong inverse correlation between tumorgraft p16 and p53 or Rb was identified (Spearman correlations P = 0.085 and P = 0.002, respectively). Significant growth inhibition of representative tumorgrafts was shown with cisplatin, cetuximab, or radiation treatment delivered over a two-week period. Early passage cell strains showed high consistency in response to cancer therapy between tumorgraft and cell strain.Conclusions: We have established a robust human tumorgraft model system for investigating HPV-positive and HPV-negative HNSCC. These tumorgrafts show strong correlation with the original tumor specimens and provide a powerful resource for investigating mechanisms of therapeutic response as well as preclinical testing. Clin Cancer Res; 19(4); 855–64. ©2012 AACR.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1−/− accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1−/− (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

Chronic myeloid leukemia (CML) is defined by the presence of Philadelphia chromosome (Ph) which results from a reciprocal translocation between chromosomes 9 and 22 [t(9;22] that gives rise to aBCR-ABL1fusion gene. CML occurs in 3 different phases (chronic, accelerated, and blast phase) and is usually diagnosed in the chronic phase. Tyrosine kinase inhibitor therapy is a highly effective first-line treatment option for all patients with newly diagnosed chronic phase CML. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with chronic phase CML.

Diffuse large B-cell lymphoma featuring overexpression of MYC and B-Cell Lymphoma 2 (double expressor lymphoma, DEL) is associated with poor outcomes. Existing evidence suggesting improved outcomes for DEL with the use of more intensive regimens than R-CHOP is restricted to younger patients and based on limited evidence from low patient numbers. We retrospectively evaluated the impact of intensive frontline regimens versus R-CHOP in a multicenter analysis across 7 academic medical centers in the United States. We collected 90 cases of DEL, 46 out of 90 patients (51%) received R-CHOP and 44/90 (49%) received an intensive regimen, which was predominantly DA-EPOCH-R. Treatment cohorts were evenly balanced for demographics and disease characteristics, though the intensive group had a higher lactate dehydrogenase (LDH, 326 vs. 230 U/L p = 0.06) and presence of B-symptoms (50% vs. 22%, p = 0.01) compared to the R-CHOP cohort. There was no difference in PFS (median 53 vs. 38 months, p = 0.49) or overall survival (67 vs. not reached months, p = 0.14) between the R-CHOP and intensive therapy cohorts, respectively. On multivariate analysis, intensive therapy was associated with a hazard ratio of 2.35 (95% CI 0.74-7.41), though this was not statistically significant. Additionally, a subgroup analysis of intermediate high-risk lymphoma defined by IPI ≥3 did not identify a difference in survival outcomes between regimens. We conclude that in our multi-center cohort there is no evidence supporting the use of intensive regimens over R-CHOP, suggesting that R-CHOP remains the standard of care for treating DEL.

Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell–like subtype (ABC) than germinal center B cell–like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL. Implications: The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited nonmalignant lymphoproliferative disorder characterized by heterozygous mutations within the first apoptosis signal receptor (FAS) signaling pathway. Defects in FAS-mediated apoptosis cause an expansion and accumulation of autoreactive CD4− and CD8− (double-negative) T cells, leading to cytopenias, splenomegaly, lymphadenopathy, autoimmune disorders, and a greatly increased lifetime risk of lymphoma. The differential diagnosis of ALPS includes infection, other inherited immunodeficiency disorders, primary and secondary autoimmune syndromes, and lymphoma. The most consistent pathologic feature is a florid paracortical expansion of double-negative T cells in lymph nodes. A presumptive clinical diagnosis can be made from symptoms and a constellation of laboratory test results. However, a definitive diagnosis requires ancillary testing and enables disease subclassification. Recognition of ALPS is critical, as treatment with immunosuppressive therapies can effectively reduce or ameliorate symptoms for most patients.

We and others have previously shown that KrasG12D is a much more potent oncogene than oncogenic Nras in hematological malignancies. We attributed the strong leukemogenic activity of KrasG12D at least partially to its unique capability to hyperactivate wild-type (WT) Nras and Hras. Here, we report that Sos1, a guanine nucleotide exchange factor, is required to mediate this process. Sos1 is overexpressed in KrasG12D/+ cells, but not in NrasQ61R/+ and NrasG12D/+ cells. KrasG12D proteins form a complex with Sos1 in vivo. Sos1 deficiency attenuates hyperactivation of WT Nras, Hras, and the downstream ERK signaling in KrasG12D/+ cells. Thus, Sos1 deletion ameliorates oncogenic Kras-induced myeloproliferative neoplasm (MPN) phenotypes and prolongs the survival of KrasG12D/+ mice. In contrast, Sos1 is dispensable for hyperactivated granulocyte-macrophage colony-stimulating factor signaling in NrasQ61R/+ cells, and Sos1−/− does not affect MPN phenotypes in NrasQ61R/+ mice. Moreover, the survival of KrasG12D/+; Sos1−/− recipients is comparable to that of KrasG12D/+ recipients treated with combined MEK and JAK inhibitors. Our study suggests that targeting Sos1-oncogenic Kras interaction may improve the survival of cancer patients with KRAS mutations.

Autoimmune lymphoproliferative syndrome (ALPS) is an autoimmune disorder characterized by a polyclonal lymphoproliferation that leads to lymphadenopathy, splenomegaly, and autoimmune cytopenias. Most cases present in pediatric patients due to inherited germline mutations affecting the FAS-mediated apoptosis pathway that mediates development of the immune system, particularly the elimination of autoreactive T cells. It is a rare disease that clinicians need to recognize in order to appropriately pursue additional ancillary testing that includes screening for circulating double-negative T cells, elevated cytokine levels, characteristic histologic findings on lymph node biopsies, and definitive follow-up evaluation with gene sequencing. Management of ALPS centers around selecting the appropriate immunosuppressive regimen to treat autoimmune cytopenias, avoiding splenectomy, and long-term surveillance for lymphomatous transformation.

Early reports have raised concerns regarding the clinical sensitivity of nasopharyngeal SARS-CoV-2 reverse transcriptase-polymerase chain reaction (RT-PCR) testing for patients with COVID-19 symptoms, which has led to requests for repeat testing at our institution. However, to our knowledge, there are no reports to date of the utilization or results of repeat testing to help guide this practice.The authors searched the institutional laboratory information system for consecutive patients who were tested for SARS-CoV-2 by RT-PCR of a nasopharyngeal specimen over a 1-month period. Characteristics and results of patients who received a single or multiple tests were documented and analyzed.Six thousand three (6003) tests were performed on 5757 patients; 272 (4.7%) patients were positive based on their initial test results. Two hundred thirty-six (4%) patients were tested more than once, with 226 (96%) tested twice. The largest proportion of these patients (n=160, 71%) were those who had an initial negative test followed by a repeat test for persistent symptoms. This group included all 7 patients who had discordant positive results on their second test; the result concordance rate within this group was 96%.In a population of patients with a low positive rate for SARS-CoV-2 by nasopharyngeal RT-PCR testing, repeat nasopharyngeal testing of negative patients who have persistent symptoms still yields a negative result in 96% of the cases.

Non-Hodgkin lymphomas (NHL) are a biologically and clinically heterogeneous collection of lymphoid malignancies. The majority of NHLs are of B-cell origin, with a minority deriving from T cells. The most common B-cell NHLs include diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphomas, and mantle cell lymphoma. The most common T-cell lymphoma is peripheral T-cell lymphoma not otherwise specified, followed by anaplastic large cell lymphomas and angioimmunoblastic T-cell lymphoma. Accurate diagnosis requires histologic evaluation and immunophenotyping in most cases, with certain subtypes requiring genetic/molecular diagnostics. Most lymphomas are sporadic, though risk is increased in the setting of immunodeficiency, autoimmune disease, and infection with certain oncogenic viruses. Staging evaluation is subtype specific, but usually requires a f PET/CT scan and may or may not require bone marrow evaluation. Prognosis is dependent on NHL subtype. Aggressive histologies are potentially curable, but have a life expectancy typically measured in weeks to months without effective therapy. Indolent histologies are usually considered incurable with conventional therapies, but are highly manageable with a typical prognosis measured in decades and most patients having a normal life expectancy. Anti-CD20 monoclonal antibodies combined with chemotherapy remains the standard initial management of most B-cell lymphomas, while chemotherapy alone remains standard in T-cell lymphomas. Numerous novel treatments are available for relapsed/refractory NHL subtypes including lenalidomide, BTK inhibitors, PI3 Kinase inhibitors, and anti-CD19 CAR T-cells. Novel agents are undergoing extensive evaluation in the upfront setting and will likely add to or replace chemotherapy in selected NHL subtypes in the future.

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling significantly upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia.

Chronic myeloid leukemia (CML) is defined by the presence of Philadelphia chromosome (Ph), resulting from a reciprocal translocation between chromosomes 9 and 22 [t(9;22] that gives rise to a BCR-ABL1 fusion gene. CML occurs in 3 different phases (chronic, accelerated, and blast phase) and is usually diagnosed in the chronic phase. Tyrosine kinase inhibitor (TKI) therapy is a highly effective first-line treatment option for all patients with newly diagnosed chronic phase CML (CP-CML). The selection TKI therapy should be based on the risk score, toxicity profile of TKI, patient's age, ability to tolerate therapy, and the presence of comorbid conditions. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with CP-CML.

7503 Background: Optimal initial therapy for mantle cell lymphoma (MCL) remains uncertain. The randomized phase 2 NCTN trial E1411 tested if progression-free survival (PFS) is prolonged by addition of bortezomib (V) (1.6 mg/m2 SC/IV days 1, 8) to bendamustine-rituximab (BVR vs BR) induction and/or by addition of lenalidomide (L) to rituximab (LR vs R) consolidation. Here we report efficacy and toxicity of induction BVR vs BR. Methods: 373 pts, accrued 2012–16, stratified by MIPI and age (≥60) received 1 of 4 arms: A) BR induction x 6 followed by R x 2 yrs, B) BVR followed by R, C) BR followed by LR or D) BVR followed by LR. Eligible pts had untreated MCL, ≥ age 18 (amended from ≥60 when S1106 for < 65 closed), ECOG PS 0-2 and adequate hematologic and organ function. Pts without progressive disease during induction proceeded to consolidation. Primary induction objective was whether adding bortezomib (BVR) (Arms B + D) to BR (Arms A + C) improves PFS, irrespective of consolidation R vs LR. Design of 360 eligible treated pts would provide 93.8% power to detect 10% improvement in 2-yr PFS from 70% hypothesized for BR, corresponding to 37.4% reduction in hazard using stratified log-rank test at 1-sided 10% alpha. Efficacy population was 179 (BVR) and 180 (BR), induction treatment completed in 144 vs 153, progressive disease during induction 6 vs 7 and registration to consolidation 140 vs 145. Results: Baseline demographics did not differ between the groups, with median age 67 (range 42-90) and 13% < 60 yr, 73% men, ECOG PS 0-1 97%, MIPI Low/Med/Hi 37/29/34%. Estimated PFS at 2 yrs 79.6% BVR (95% CI 73.8-85.9) vs 74.5% BR (95% CI 68.2-81.4) (1-sided stratified log-rank p = 0.268). With median PFS follow-up 51 mos, median PFS estimated at 64.1 and 64.0 mos. Overall response rate (ORR) for BVR was 88.9% (CR 65.5%) vs 89.5% (CR 60.5%) BR (z-test 1 sided p = 0.577 for ORR). Treatment related deaths during induction were 2 in BVR (cardiac arrest, hepatitis) and 1 in BR (tumor lysis). Grade ≥ 3 toxicities were 88.1% (163/185) BVR vs 77.5% (145/187) BR. For BVR vs BR grade ≥ 3 neutropenia occurred in 52 vs 39 pts, though febrile neutropenia (7 vs 6), anemia (7 vs 8) and thrombocytopenia (18 vs 16) did not differ. Peripheral neuropathy (PN) grade 2 was 8 sensory for BVR vs 2 sensory/1 motor for BR, while grade 3 PN was 6 sensory/1 motor for BVR vs 0 with BR. The only non-hematologic grade ≥ 3 toxicity in > 5% of pts was rash (9 vs 12 pts). Conclusions: Bortezomib did not significantly improve the primary endpoint of PFS when added to BR as initial MCL therapy. ORR and CR rates at end of induction were also similar. Follow-up continues to assess the entire treatment regimen, including consolidation R vs LR, but the PFS > 5 yrs, high ORR and MRD negativity rate (Smith et al ASH 2019) in this BR-based trial support BR as a platform for MCL induction therapy. Clinical trial information: NCT01415752.

We report a unique case of composite hairy cell leukemia (HCL) and monoclonal B-cell lymphocytosis with chronic lymphocytic leukemia (CLL) phenotype evaluated comprehensively through cell sorting and deep sequencing. The patient presented with decreased exercise tolerance and complete blood count revealed neutropenia, monocytopenia, and thrombocytopenia. The peripheral blood film was suggestive of HCL. However, a bone marrow evaluation was suspicious for composite HCL and CLL. Flow cytometry not only confirmed monoclonal kappa light chain restricted HCL and CLL populations, but also identified a kappa restricted population with co-expression of bright CD20, bright CD22, and CD11c without CD25 or CD103. Each population was isolated by cell sorting and subsequent B-cell receptor gene rearrangement analysis showed distinct rearrangements in each population. Likewise, next-generation sequencing (NGS) showed distinct mutation patterns in each of the monoclonal B-cell populations. The HCL clone harbored the signature BRAF V600E mutation, the CLL clone harbored an RB1 (L343fs*6) mutation, and the third clone was essentially negative for either mutation. In HCL, the BRAF V600E has been found in hematopoietic stem cells, raising the possibility of a common stem cell origin for composite HCL/CLL. However, our findings suggest a process of independent clonal development of multiple neoplastic B-cell populations in composite lymphoma, likely occurring somewhere after the common lymphoid progenitor stage.

Somatic mutations in TP53 and NRAS are associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RAS pathway and TP53 mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53 loss and endogenous NrasG12D/+ in AML, we generated conditional NrasG12D/+p53-/- mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53-/- bone marrow cells rapidly develop a highly penetrant AML. We find that p53-/- cooperates with NrasG12D/+ to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53-/- MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53 loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53-/- synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML.

Summary Rearrangement of MYC is associated with a poor prognosis in patients with diffuse large B cell lymphoma (DLBCL) and B cell lymphoma unclassifiable (BCLU), particularly in the setting of double hit lymphoma (DHL). However, little is known about outcomes of patients who demonstrate MYC rearrangement without evidence of BCL2 or BCL6 rearrangement (single hit) or amplification (>4 copies) of MYC. We identified 87 patients with single hit lymphoma (SHL), 22 patients with MYC-amplified lymphoma (MYC amp) as well as 127 DLBCL patients without MYC rearrangement or amplification (MYC normal) and 45 patients with DHL, all treated with either R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) or intensive induction therapy. For SHL and MYC amp patients, the 2-year progression-free survival rate (PFS) was 49% and 48% and 2-year overall survival rate (OS) was 59% and 71%, respectively. SHL patients receiving intensive induction experienced higher 2-year PFS (59% vs. 23%, P = 0·006) but similar 2-year OS as compared with SHL patients receiving R-CHOP. SHL DLBCL patients treated with R-CHOP, but not intensive induction, experienced significantly lower 2-year PFS and OS (P

Targeting Bruton tyrosine kinase (BTK) by ibrutinib is an effective treatment for patients with relapsed/refractory mantle cell lymphoma (MCL). However, both primary and acquired resistance to ibrutinib have developed in a significant number of these patients. A combinatory strategy targeting multiple oncogenic pathways is critical to enhance the efficacy of ibrutinib. Here, we focus on the BCL2 anti-apoptotic pathway. In a tissue microarray of 62 MCL samples, BCL2 expression positively correlated with BTK expression. Increased levels of BCL2 were shown to be due to a defect in protein degradation because of no or little expression of the E3 ubiquitin ligase FBXO10, as well as transcriptional upregulation through BTK-mediated canonical nuclear factor-κB activation. RNA-seq analysis confirmed that a set of anti-apoptotic genes (for example, BCL2, BCL-XL and DAD1) was downregulated by BTK short hairpin RNA. The downregulated genes also included those that are critical for B-cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Targeting BCL2 by the specific inhibitor ABT-199 synergized with ibrutinib in inhibiting growth of both ibrutinib-sensitive and -resistant cancer cells in vitro and in vivo. These results suggest co-targeting of BTK and BCL2 as a new therapeutic strategy in MCL, especially for patients with primary resistance to ibrutinib.

BACKGROUND Double-hit lymphomas (DHLs) are collectively defined as B-cell non-Hodgkin lymphomas harboring rearrangements of MYC as well as B-cell lymphoma 2 (BCL2) and/or B-cell lymphoma 6 (BCL6). To the authors' knowledge, the impact of specific oncogene rearrangements on outcomes of patients with DHL who are treated with immunochemotherapy has not been previously described. METHODS The authors identified patients whose diagnostic tissue specimens underwent metaphase karyotyping or fluorescence in situ hybridization for MYC as well as both BCL2 and BCL6 rearrangements. Cohorts were defined by the presence (+) or absence (−) of rearrangements: MYC+/BCL2+/BCL6− (BCL2-DHL), MYC+/BCL2−/BCL6+ (BCL6-DHL), and MYC+/BCL2+/BCL6+ (triple-hit lymphoma; THL). RESULTS A total of 117 patients were included in the current analysis (76 BCL2-DHL patients, 16 BCL6-DHL patients, and 25 THL patients). Compared with patients with BCL2-DHL, those with BCL6-DHL were more likely to be classified as having a non-germinal center cell of origin, presented with extranodal disease, and appeared to achieve higher rates of complete response despite receiving intensive induction therapy less frequently. However, patients with BCL6-DHL experienced a shorter median overall survival if achieving an initial complete response compared with patients with BCL2-DHL. Patients with THL experienced survival outcomes similar to those of patients with BCL2-DHL. CONCLUSIONS Recognition of the specific oncogene rearrangements may be of prognostic value and potentially guide future therapeutic strategies for patients with DHL. Cancer 2016;122:559–564. © 2015 American Cancer Society.

Summary: Metabolic flexibility allows cells to adapt to various environments and limits the efficacy of metabolic drugs. Therapeutic targeting of cancer metabolism relies on defining limiting requirements and vulnerabilities in the highly dynamic metabolic network. Here, we characterize the metabolic reprogramming and identify cancer-specific metabolic vulnerabilities in response to the pharmacological inhibition of mitochondrial complex I. Our work reveals the adaptation mechanism in malignant lymphocytes providing resistance against the biguanides phenformin and metformin by transcriptionally reprogramming glucose metabolism. Metabolic adaptation to complex I inhibition is mediated by mitochondrial reactive oxygen species (mROS) serving as a mitochondrial stress signal activating hypoxia-inducible factor-1a (HIF-1a). Inhibition of the mROS/HIF-1a axis through antioxidants or direct suppression of HIF-1a selectively disrupts metabolic adaptation and survival during complex I dysfunction in malignant lymphocytes. Our results identify HIF-1a signaling as a critical factor in resistance against biguanide-induced mitochondrial dysfunction, allowing selective targeting of metabolic pathways in leukemia and lymphoma. : Khan et al. characterize the signaling mechanisms underlying metabolic adaptation and fuel utilization of malignant lymphocytes in response to mitochondrial dysfunction induced by biguanides. Suppression of HIF-1a selectively targets malignant lymphocytes by inducing synthetic lethality in response to metformin and phenformin. Keywords: metabolic flexibility, biguanides, mitochondria, HIF-1a, lymphoid malignancies

INTRODUCTION Bone marrow biopsy (BMB) is crucial for the diagnosis, staging, and monitoring of a variety of hematologic diseases. Obtaining an adequate BMB can be challenging given the need to balance patient comfort with acquisition of high quality specimens. We had observed variable BMB quality at our institution with poor quality specimens sometimes affecting diagnosis. We thus undertook this quality improvement (QI) project to improve the quality of diagnostic BMB specimens. METHODS We used an A3 QI process to identify factors possibly influencing BMB quality. We collected baseline data on 211 BMB, with short and long-term follow-up data on a further 382 cases. We used clinical conferences to discuss data, perform peer comparisons and identify strategies to create a sustainable improvement in BMB quality. RESULTS Baseline data showed that BMB length was influenced most by the individual performer, with some influence of needle gauge. Other factors such as sedation, BMB indication were noncontributory. BMB lengths improved following performer education and individual performer data comparisons (15.2 mm post vs 12.8 mm baseline, P

ObjectiveDespite improvements in surgery and chemotherapy, ovarian cancer remains a deadly disease in need of improved therapies. We have previously shown that Notch1 intracellular domain (NICD) is highly expressed in ovarian cancer. We have also shown that NICD inhibition can lead to growth arrest in ovarian cancer cells. The objective of the current study was to delineate whether NICD expression correlates with prognosis of women with ovarian cancer.MethodsAfter the institutional review board approval, patients with a diagnosis of primary ovarian cancer between the years 2001 and 2007 who underwent surgery at our institution were identified. Paraffin blocks from the primary ovarian tumor were analyzed, and core samples were obtained to build a tissue microarray. Cytoplasmic NICD expression was assessed by quantitative immunofluorescent morphometry using the automated quantitative analysis system. These results were correlated with clinical and pathology data retrieved from the patient records.ResultsWe identified 328 patients with primary ovarian cancer during this period. Seventeen percent of patients had stage I, 11% had stage II, 59% had stage III, and 13% had stage IV disease. Most patients (70%) had papillary serous histology, and most (86%) underwent optimal debulking to less than 1 cm of residual disease. High NICD expression was found to correlate strongly with low overall survival (P = 0.001). This effect remained in multivariate analysis (P = 0.023).ConclusionsHigh expression of NICD in the primary tumor of women with ovarian cancer is an independently poor prognostic factor for overall survival. Further research into the therapeutic inhibition of the Notch1 pathway is warranted.

Human pegivirus (HPgV) protects HIV+ people from HIV-associated disease, but the mechanism of this protective effect remains poorly understood. We sequentially infected cynomolgus macaques with simian pegivirus (SPgV) and simian immunodeficiency virus (SIV) to model HIV+HPgV co-infection. SPgV had no effect on acute-phase SIV pathogenesis – as measured by SIV viral load, CD4+ T cell destruction, and immune activation – suggesting that HPgV’s protective effect is exerted primarily during the chronic phase of HIV infection. We also examined the immune response to SPgV in unprecedented detail, and found that this virus elicits virtually no activation of the immune system despite persistently high titers in the blood over long periods of time. Overall, this study expands our understanding of the pegiviruses – an understudied group of viruses with a high prevalence in the global human population – and suggests that the protective effect observed in HIV+HPgV co-infected people occurs primarily during the chronic phase of HIV infection.One Sentence SummaryPegivirus avoids immune recognition but does not attenuate acute-phase disease in a macaque model of HIV infection.Short TitlePegivirus and AIDS-virus co-infectionAccessible SummaryPeople infected with HIV live longer, healthier lives when they are co-infected with the human pegivirus (HPgV) – an understudied virus with a high prevalence in the global human population. To better understand how HPgV protects people with HIV from HIV-associated disease, we infected macaques with simian versions of these two viruses (SPgV and SIV). We found that SPgV had no impact on the incidence of SIV-associated disease early during the course of SIV infection – a time when SIV and HIV are known to cause irreversible damage to the immune system. Oddly, we found that the immune system did not recognize SPgV; a finding that warrants further investigation. Overall, this study greatly expands on our understanding of the pegiviruses and their interaction with the immune system.

Introduction VcR-CVAD was developed as an intermediate-intensity induction regimen with maintenance rituximab (MR) to improve remission durations after first-line therapy for mantle cell lymphoma (MCL) in older and younger patients with MCL. Patients and Methods Patients with previously untreated MCL received VcR-CVAD induction chemotherapy for 6 cycles (21-day cycles). Patients achieving at least a partial response received rituximab consolidation (375 mg/m2 × 4 weekly doses) and MR (375 mg/m2 every 12 weeks × 20 doses). The primary endpoints were overall and complete response (CR), and the secondary endpoints were progression-free survival (PFS) and overall survival (OS). Thirty patients were enrolled, with a median age of 61 years. There was an even distribution of patients Conclusions Long-term outcomes with VcR-CVAD are comparable with more intensive inductions and consolidation approaches. MCL is biologically heterogeneous, and durable remission can be achieved with intermediate intensity therapy. MR appears to contribute to these excellent outcomes.

Objective: One prospective treatment option for vocal fold scarring is regeneration with an engineered scaffold containing induced pluripotent stem cells (iPS). In the present study, we investigated the feasibility of utilizing an injectable hyaluronic acid (HA) scaffold encapsulated with human-iPS cell (hiPS) for regeneration of vocal folds. Methods: Thirty athymic nude rats underwent unilateral vocal fold injury. Contralateral vocal folds served as uninjured controls. Hyaluronic acid hydrogel scaffold, HA hydrogel scaffold containing hiPS, and HA hydrogel scaffold containing hiPS with epidermal growth factor (EGF) were injected in both vocal folds immediately after surgery. One and 2 weeks after injection, larynges were excised for histology, immunohistochemistry, and fluorescence in situ hybridization (FISH). Results: Presence of HA hydrogel was confirmed in vocal folds 1 and 2 weeks post injection. The FISH analysis confirmed the presence and viability of hiPS in the injected vocal folds. Histological results demonstrated that vocal folds injected with HA hydrogel scaffold containing EGF demonstrated less fibrosis than those with HA hydrogel only. Conclusions: Human-iPS survived in injured rat vocal folds. The HA hydrogel with hiPS and EGF ameliorated the fibrotic response. Additional work is necessary to optimize hiPS differentiation and further confirm the safety of hiPS for clinical applications.

Objectives: Flow cytometry is essential for the evaluation of lymphoproliferative disorders (LPDs) and their classification. Flow panels routinely incorporate a large array of antibodies, making testing complex and expensive; such panels are likely unnecessary in benign cases or those with straightforward diagnoses. Our aim was to develop a more cost-effective testing strategy based on a retrospective analysis of flow studies for possible LPDs in blood. Methods: We identified LPD frequencies and types, as well as associated results with patient age and absolute lymphocyte count. Results: We found that the likelihood of LPDs increased with patient age and absolute lymphocyte count and that CD5-positive LPD was the most common LPD diagnosed in our institution (71% of LPDs). Using these data, we devised flow-testing algorithms with a screening test for patients at low risk of disease and a focus on CD5-positive LPD detection, with reflexing as needed. Conclusions: We project this approach will result in a 40% decrease in antibody utilization.

Objective An intact epithelium is an important part of vocal fold defense. Damage to the epithelium can compromise vocal fold homeostasis and protection of the host tissue from viral and bacterial invasion. Elucidating the effects of damage on epithelial architectural and barrier integrity provides insight into the role of epithelium in protecting vocal folds. Using an animal model, we evaluated the time course of structural and functional epithelial restoration following injury.

Targeted modulation of microenvironmental regulatory pathways may be essential to control myeloma and other genetically/clonally heterogeneous cancers. Here we report that human myeloma-associated monocytes/macrophages (MAM), but not myeloma plasma cells, constitute the predominant source of interleukin-1β (IL-1β), IL-10, and tumor necrosis factor-α at diagnosis, whereas IL-6 originates from stromal cells and macrophages. To dissect MAM activation/cytokine pathways, we analyzed Toll-like receptor (TLR) expression in human myeloma CD14(+) cells. We observed coregulation of TLR2 and TLR6 expression correlating with local processing of versican, a proteoglycan TLR2/6 agonist linked to carcinoma progression. Versican has not been mechanistically implicated in myeloma pathogenesis. We hypothesized that the most readily accessible target in the versican-TLR2/6 pathway would be the mitogen-activated protein 3 (MAP3) kinase, TPL2 (Cot/MAP3K8). Ablation of Tpl2 in the genetically engineered in vivo myeloma model, Vκ*MYC, led to prolonged disease latency associated with plasma cell growth defect. Tpl2 loss abrogated the "inflammatory switch" in MAM within nascent myeloma lesions and licensed macrophage repolarization in established tumors. MYC activation/expression in plasma cells was independent of Tpl2 activity. Pharmacologic TPL2 inhibition in human monocytes led to dose-dependent attenuation of IL-1β induction/secretion in response to TLR2 stimulation. Our results highlight a TLR2/6-dependent TPL2 pathway as novel therapeutic target acting nonautonomously through macrophages to control myeloma progression.

Overactive RAS signaling is prevalent in juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) in humans, and both are refractory to conventional chemotherapy. Conditional activation of a constitutively active oncogenic Nras (NrasG12D/G12D) in murine hematopoietic cells promotes an acute myeloproliferative neoplasm (MPN) that recapitulates many features of JMML and MP-CMML. We found that NrasG12D/G12D-expressing HSCs, which serve as JMML/MP-CMML-initiating cells, show strong hyperactivation of ERK1/2, promoting hyperproliferation and depletion of HSCs and expansion of downstream progenitors. Inhibition of the MEK pathway alone prolonged the presence of NrasG12D/G12D-expressing HSCs but failed to restore their proper function. Consequently, approximately 60% of NrasG12D/G12D mice treated with MEK inhibitor alone died within 20 weeks, and the remaining animals continued to display JMML/MP-CMML-like phenotypes. In contrast, combined inhibition of MEK and JAK/STAT signaling, which is commonly hyperactivated in human and mouse CMML, potently inhibited human and mouse CMML cell growth in vitro, rescued mutant NrasG12D/G12D-expressing HSC function in vivo, and promoted long-term survival without evident disease manifestation in NrasG12D/G12D animals. These results provide a strong rationale for further exploration of combined targeting of MEK/ERK and JAK/STAT in treating patients with JMML and MP-CMML.

Triple-negative breast cancer (TNBC) is a subclass of breast cancers (i.e., estrogen receptor–negative, progesterone receptor–negative, and HER2-negative) that have poor prognosis and very few identified molecular targets. Strikingly, a high percentage of TNBCs overexpresses the EGF receptor (EGFR), yet EGFR inhibition has yielded little clinical benefit. Over the last decade, advances in EGFR biology have established that EGFR functions in two distinct signaling pathways: (i) classical membrane-bound signaling and (ii) nuclear signaling. Previous studies have demonstrated that nuclear EGFR (nEGFR) can enhance resistance to anti-EGFR therapies and is correlated with poor overall survival in breast cancer. On the basis of these findings, we hypothesized that nEGFR may promote intrinsic resistance to cetuximab in TNBC. To examine this question, a battery of TNBC cell lines and human tumors were screened and found to express nEGFR. Knockdown of EGFR expression demonstrated that TNBC cell lines retained dependency on EGFR for proliferation, yet all cell lines were resistant to cetuximab. Furthermore, Src Family Kinases (SFKs) influenced nEGFR translocation in TNBC cell lines and in vivo tumor models, where inhibition of SFK activity led to potent reductions in nEGFR expression. Inhibition of nEGFR translocation led to a subsequent accumulation of EGFR on the plasma membrane, which greatly enhanced sensitivity of TNBC cells to cetuximab. Collectively, these data suggest that targeting both the nEGFR signaling pathway, through the inhibition of its nuclear transport, and the classical EGFR signaling pathway with cetuximab may be a viable approach for the treatment of patients with TNBC. Mol Cancer Ther; 13(5); 1356–68. ©2014 AACR.

Sex determining region Y-box 11 (SOX11) expression is specific for mantle cell lymphoma (MCL) as compared with other non-Hodgkin's lymphomas. However, the function and direct-binding targets of SOX11 in MCL are largely unknown. We used high-resolution chromatin immunoprecipitation sequencing to identify the direct target genes of SOX11 in a genome-wide, unbiased manner and elucidate its functional significance. Pathway analysis identified WNT, PKA and TGF-beta signaling pathways as significantly enriched by SOX11-target genes. Quantitative chromatin immunoprecipitation sequencing and promoter reporter assays confirmed that SOX11 directly binds to individual genes and modulates their transcription activities in these pathways in MCL. Functional studies using RNA interference demonstrate that SOX11 directly regulates WNT in MCL. We analyzed SOX11 expression in three independent well-annotated tissue microarrays from the University of Wisconsin (UW), Karolinska Institute and British Columbia Cancer Agency. Our findings suggest that high SOX11 expression is associated with improved survival in a subset of MCL patients, particularly those treated with intensive chemotherapy. Transcriptional regulation of WNT and other biological pathways affected by SOX11-target genes may help explain the impact of SOX11 expression on patient outcomes.

The NCCN Guidelines for Chronic Myeloid Leukemia (CML) provide recommendations for the management of chronic-phase and advanced-phase CML in adult patients. The median age of disease onset is 67 years. However, because CML occurs in all age groups, clinical care teams should be prepared to address issues relating to fertility and pregnancy with patients who are of reproductive age at the time of diagnosis. CML is relatively rare in children and there are no evidence-based recommendations for the management of CML in pediatric population. These NCCN Guidelines Insights discuss special considerations for the management of CML during pregnancy and for the management of CML in the pediatric population.

In the original version of [this article][1] ([1][2]), three figures are incorrect. Fig. 6 uses the same image to represent “No Antibody” for UW-SCC36 and UW-SCC6; Fig. 2C includes bar graphs that exhibit irregularities, including an improperly scaled y -axis; and Fig. 2B contains duplicate

Introduction: Genetic rearrangements in both the MYC gene and anti-apoptotic genes such as BCL2 or BCL6 in diffuse large B-cell lymphoma (DLBCL) represent a sub-type of lymphoma, termed "double-hit lymphoma" (DHL) that is associated with poor outcomes and a poor response to standard chemoimmunotherapy regimens such as rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) (Petrich et al, Blood, 2014). The incidence of DHL is low at approximately 5-10% of DLBCL presentations. Alternatively, DLBCL cases featuring dual expression of both MYC and BCL-2 and/or BCL-6 (as detected by immunohistochemistry [IHC]) occur more frequently with increasing evidence to suggest this lymphoma-subtype, termed "double-expressor lymphoma" (DEL) may also respond poorly to induction therapy with R-CHOP (Green et al, JCO, 2012). Studies examining outcomes for DEL are often limited by an inability to separate DHL from DEL or lack a direct comparison to R-CHOP and, as a result, optimal induction therapy for DEL remains an unanswered question. Methods We performed a retrospective review of outcomes in DEL according to initial treatment regimen (R-CHOP vs intensive therapy) for patients diagnosed from 2013-2016 at the University of Wisconsin. We excluded patients lacking IHC or FISH data for MYC or BCL-2, those receiving a non-anthracycline containing regimen, those with concurrent central nervous system presentation, and those with post-transplant lymphoproliferative disorder. Any patients meeting criteria for DHL (i.e. dual gene rearrangements) were coded as DHL and not coded as DEL regardless of MYC, BCL-2, or BCL-6 expression. Intensive therapy included dose adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R), or rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone (R-hyper-CVAD). Results From 200 cases of DLBCL, we identified 31 (16%) cases of DEL and 11 (6%) cases of DHL. Twenty-five cases of DEL and 9 cases of DHL were eligible for inclusion in our study. Characteristics were similar between the DHL and DEL cohort, although there were more cases of early stage disease in the DEL cohort (56% vs 11%, P=0.05). For DEL, the median age for the R-CHOP cohort was older compared to the intensive cohort (table 1, 73 vs 63), but both groups had a similar percentage of patients aged >60 years (67 vs 80%, P=0.66). Otherwise, demographics including performance status, stage, and revised international prognostic index score were well balanced between the two treatment groups with the exception of more cases of germinal center B-cell of origin (GCB) within the R-CHOP group (80 v 30%, P=0.03). Complete response rates to induction regimens for DEL were similar between the R-CHOP and intensive group (76 vs 80%, P=0.65). Median progression free survival (PFS) was extended in the intensive cohort although 2 year PFS rates were similar between the two groups (40 vs 50%, P=0.70). Two-year overall survival rates were also similar (80 vs 70%, P=0.65). Kaplan-Meier survival curves (figure 1) illustrate a potential benefit for intensive induction therapy, however this is not statistically significant (hazard ratio 0.69 P=0.65). Compared to patients with DEL, patients with DHL achieved similar complete response rates to induction therapy (76% vs 67%, P=0.67) despite exclusive use of DA-EPOCH-R for all DHL patients. There were more consolidative autologous stem cell transplants (auto-SCT) performed in the DHL group (33% vs 8%, P=0.1). PFS and OS were also similar between DHL and DEL sub-groups (HR 1.09, P=0.91) Conclusions Our study is amongst the first to compare R-CHOP to more intensive regimens for DEL while also separating DEL from DHL. Our current sample size limits our ability to draw definitive conclusions regarding outcomes by treatment regimen; however, despite lack of statistical significance, there is a trend toward intensive regimens improving PFS and OS in DEL. Our study is also amongst the first to directly compare outcomes for DEL and DHL. Survival for DEL was similar to DHL and may reflect improvements in outcomes for DHL when using intensive regimens or may be related to the increased use of consolidative auto-SCT for DHL. Our sample is part of a planned larger multi-center analysis and an increased sample size may build on the improved survival trend demonstrated for intensive regimens vs R-CHOP observed in our analysis. Disclosures No relevant conflicts of interest to declare.

Cobalamin (vitamin B12) deficiency is a common cause of megaloblastic anemia in Western populations. Laboratory evaluation of megaloblastic anemia frequently includes the assessment of patient cobalamin and folate status. Current total serum cobalamin measurements are performed in the clinical laboratory with competitive binding luminescence assays, whose results may not always accurately reflect actual cobalamin stores. Surrogate markers of cobalamin deficiency such as methylmalonic acid and homocysteine have been utilized to improve diagnostic accuracy; however, the specificity of these tests by themselves is rather low. Measurement of the biologically active fraction of cobalamin, holotranscobalamin, has been proposed as a replacement for current total cobalamin assays. Although holotranscobalamin measurements appear to have slighter better sensitivity, the specificity of this assay remains to be determined. The relative merits and demerits of commonly available methods to assess cobalamin deficiency in patients with suspected megaloblastic anemia are discussed.

Since the highly conserved exosome complex mediates the degradation and processing of multiple classes of RNAs, it almost certainly controls diverse biological processes. How this post-transcriptional RNA-regulatory machine impacts cell fate decisions and differentiation is poorly understood. Previously, we demonstrated that exosome complex subunits confer an erythroid maturation barricade, and the erythroid transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the cognate genes. While dissecting requirements for the maturation barricade in Mus musculus, we discovered that the exosome complex is a vital determinant of a developmental signaling transition that dictates proliferation/amplification versus differentiation. Exosome complex integrity in erythroid precursor cells ensures Kit receptor tyrosine kinase expression and stem cell factor/Kit signaling, while preventing responsiveness to erythropoietin-instigated signals that promote differentiation. Functioning as a gatekeeper of this developmental signaling transition, the exosome complex controls the massive production of erythroid cells that ensures organismal survival in homeostatic and stress contexts. DOI: http://dx.doi.org/10.7554/eLife.17877.001, eLife digest Red blood cells supply an animal’s tissues with the oxygen they need to survive. These cells circulate for a certain amount of time before they die. To replenish the red blood cells that are lost, first a protein called stem cell factor (SCF) instructs stem cells and precursor cells to proliferate, and a second protein, known as erythropoietin, then signals to these cells to differentiate into mature red blood cells. It is important to maintain this balance between these two processes because too much proliferation can lead to cancer while too much differentiation will exhaust the supply of stem cells. Previous work has shown that a collection of proteins called the exosome complex can block steps leading towards mature red blood cells. The exosome complex controls several processes within cells by modifying or degrading a variety of messenger RNAs, the molecules that serve as intermediates between DNA and protein. However, it was not clear how the exosome complex sets up the differentiation block and whether it is somehow connected to the signaling from SCF and erythropoietin. McIver et al. set out to address this issue by isolating precursor cells with the potential to become red blood cells from mouse fetal livers and experimentally reducing the levels of the exosome complex. The experiments showed that these cells were no longer able to respond when treated with SCF in culture, whereas the control cells responded as normal. Further experiments showed that cells with less of the exosome complex also made less of a protein named Kit. Normally, SCF interacts with Kit to instruct cells to multiply. Lastly, although the experimental cells could no longer respond to these proliferation signals, they could react to erythropoietin, which promotes differentiation. Thus, normal levels of the exosome complex keep the delicate balance between proliferation and differentiation, which is crucial to the development of red blood cells. In future, it will be important to study the exosome complex in living mice and in human cells, and to see whether it also controls other signaling pathways. Furthermore, it is worth exploring whether this new knowledge can help efforts to produce red blood cells on an industrial scale, which could then be used to treat patients with conditions such as anemia. DOI: http://dx.doi.org/10.7554/eLife.17877.002

NOTCH2 mutations in splenic marginal zone lymphoma are associated with poor prognosis., Splenic marginal zone lymphoma (SMZL), the most common primary lymphoma of spleen, is poorly understood at the genetic level. In this study, using whole-genome DNA sequencing (WGS) and confirmation by Sanger sequencing, we observed mutations identified in several genes not previously known to be recurrently altered in SMZL. In particular, we identified recurrent somatic gain-of-function mutations in NOTCH2, a gene encoding a protein required for marginal zone B cell development, in 25 of 99 (∼25%) cases of SMZL and in 1 of 19 (∼5%) cases of nonsplenic MZLs. These mutations clustered near the C-terminal proline/glutamate/serine/threonine (PEST)-rich domain, resulting in protein truncation or, rarely, were nonsynonymous substitutions affecting the extracellular heterodimerization domain (HD). NOTCH2 mutations were not present in other B cell lymphomas and leukemias, such as chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; n = 15), mantle cell lymphoma (MCL; n = 15), low-grade follicular lymphoma (FL; n = 44), hairy cell leukemia (HCL; n = 15), and reactive lymphoid hyperplasia (n = 14). NOTCH2 mutations were associated with adverse clinical outcomes (relapse, histological transformation, and/or death) among SMZL patients (P = 0.002). These results suggest that NOTCH2 mutations play a role in the pathogenesis and progression of SMZL and are associated with a poor prognosis.

An important problem in translational cancer research is our limited ability to functionally characterize behaviors of primary patient cancer cells and associated stromal cell types, and relate mechanistic understanding to therapy selection. Functional analyses of primary samples face at least 3 major challenges: limited availability of primary samples for testing, paucity of functional information extracted from samples, and lack of functional methods accessible to many researchers. We developed a microscale cell culture platform that overcomes these limitations, especially for hematologic cancers. A key feature of the platform is the ability to compartmentalize small populations of adherent and nonadherent cells in controlled microenvironments that can better reflect physiological conditions and enable cell-cell interaction studies. Custom image analysis was developed to measure cell viability and protein subcellular localizations in single cells to provide insights into heterogeneity of cellular responses. We validated our platform by assessing viability and nuclear translocations of NF-κB and STAT3 in multiple myeloma cells exposed to different conditions, including cocultured bone marrow stromal cells. We further assessed its utility by analyzing NF-κB activation in a primary chronic lymphocytic leukemia patient sample. Our platform can be applied to myriad biological questions, enabling high-content functional cytomics of primary hematologic malignancies.

Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma in the Western hemisphere. After decades of stagnation, the natural history of FL appears to have been favorably impacted by the introduction of rituximab. Randomized clinical trials have demonstrated that the addition of rituximab to standard chemotherapy induction has improved the overall survival. Maintenance rituximab strategies can improve progression-free survival. Even chemotherapy platforms have changed in the past 5 years, as bendamustine combined with rituximab has rapidly become a standard frontline strategy in North America and parts of Europe. Recent discoveries have identified patients at high risk for poor outcomes to first-line therapy (m7–Follicular Lymphoma International Prognostic Index [m7-FLIPI]) and for poor outcomes after frontline therapy (National LymphoCare Study). However, several unmet needs remain, including a better ability to identify high-risk patients at diagnosis, the development of predictive biomarkers for targeted agents, and strategies to reduce the risk of transformation. The development of targeted agents, exploiting our current understanding of FL biology, is a high research priority. A multitude of novel therapies are under investigation in both the frontline and relapsed/refractory settings. It will be critical to identify the most appropriate populations for new agents and to develop validated surrogate end points, so that novel agents can be tested (and adopted, if appropriate) efficiently.

Introduction. Hodgkin's lymphoma (HL) presenting either with primary bowel involvement or with cholestasis is unusual. The combination of primary gastrointestinal HL presenting with cholestasis and ductopenia has not been previously described. Case Report. We present a case of primary gastrointestinal HL with evidence of liver involvement, but also with prominent ductopenia on liver biopsy and associated intrahepatic cholestasis. A 50-year-old man with a history of Crohn's disease presented with a bowel obstruction, for which he underwent a small bowel resection. Histology revealed HL. His course was complicated by cholestatic liver failure. A subsequent liver biopsy revealed both focal involvement by lymphoma and ductopenia, resembling vanishing bile duct syndrome (VBDS). He was treated with chemotherapy with improvement in his cholestasis, but he eventually succumbed due to further complications of his disease and treatment toxicities. Conclusion. This case of primary gastrointestinal HL associated with ductopenia does not meet classic criteria for VBDS, but the clinical presentation and pathology are suggestive of a VBDS-like paraneoplastic process. Therapies for HL in the setting of cholestatic liver failure require special consideration, but some reports of durable remissions and recovery of liver function have been reported.

Summary Intensive chemotherapy regimens are not feasible in many adults with mantle cell lymphoma (MCL). We sought to build upon our previous experience with a non-intensive regimen, modified R-hyperCVAD chemotherapy (rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone) with maintenance rituximab (MR), by the incorporation of bortezomib (VcR-CVAD) and the extension of MR beyond 2 years. Patients with previously untreated MCL received VcR-CVAD chemotherapy every 21 d for six cycles. Patients achieving at least a partial response to induction chemotherapy received rituximab consolidation (375 mg/m2 × 4 weekly doses) and MR (375 mg/m2 every 12 weeks × 20 doses). The primary end points were overall and complete response (CR), and secondary endpoints were progression-free (PFS) and overall survival (OS). Thirty patients were enrolled, with a median age of 61 years. All patients had advanced stage disease, and 60% had medium/high MCL International Prognostic Index risk factors. A CR or unconfirmed CR was achieved in 77% of patients. After a median follow-up of 42 months, the 3-year PFS and OS were 63% and 86%, respectively. The observed 3-year PFS and OS with VcR-CVAD in MCL were comparable to reported outcomes with more intensive regimens. A cooperative group trial (E1405) is attempting to replicate these promising results.

Mantle cell lymphoma (MCL) is a mostly incurable malignancy arising from naive B cells (NBCs) in the mantle zone of lymph nodes. We analyzed genomewide methylation in MCL patients with the HELP (HpaII tiny fragment Enrichment by Ligation–mediated PCR) assay and found significant aberrancy in promoter methylation patterns compared with normal NBCs. Using biologic and statistical criteria, we further identified 4 hypermethylated genes CDKN2B, MLF-1, PCDH8, and HOXD8 and 4 hypomethylated genes CD37, HDAC1, NOTCH1, and CDK5 when aberrant methylation was associated with inverse changes in mRNA levels. Immunohistochemical analysis of an independent cohort of MCL patient samples confirmed CD37 surface expression in 93% of patients, validating its selection as a target for MCL therapy. Treatment of MCL cell lines with a small modular immunopharmaceutical (CD37-SMIP) resulted in significant loss of viability in cell lines with intense surface CD37 expression. Treatment of MCL cell lines with the DNA methyltransferase inhibitor decitabine resulted in reversal of aberrant hypermethylation and synergized with the histone deacetylase inhibitor suberoylanilide hydroxamic acid in induction of the hypermethylated genes and anti-MCL cytotoxicity. Our data show prominent and aberrant promoter methylation in MCL and suggest that differentially methylated genes can be targeted for therapeutic benefit in MCL.