Yuri Fedoriw

The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells

In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer.

Expression of p16INK4a prevents cancer and promotes aging in lymphocytes

Previous authors have suggested that tumor suppressor expression promotes aging while preventing cancer, but direct experimental support for this cancer-aging hypothesis has been elusive. Here, by using somatic, tissue-specific inactivation of the p16(INK4a) tumor suppressor in murine T- or B-lymphoid progenitors, we report that ablation of p16(INK4a) can either rescue aging or promote cancer in a lineage-specific manner. Deletion of p16(INK4a) in the T lineage ameliorated several aging phenotypes, including thymic involution, decreased production of naive T cells, reduction in homeostatic T-cell proliferation, and attenuation of antigen-specific immune responses. Increased T-cell neoplasia was not observed with somatic p16(INK4a) inactivation in T cells. In contrast, B lineage-specific ablation of p16(INK4a) was associated with a markedly increased incidence of systemic, high-grade B-cell neoplasms, which limited studies of the effects of somatic p16(INK4a) ablation on B-cell aging. Together, these data show that expression of p16(INK4a) can promote aging and prevent cancer in related lymphoid progeny of a common stem cell.

Gene Profiling of Canine B-Cell Lymphoma Reveals Germinal Center and Postgerminal Center Subtypes with Different Survival Times, Modeling Human DLBCL

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, and fewer than half of patients are cured with standard first-line therapy. To improve therapeutic options, better animal models that accurately mimic human DLBCL (hDLBCL) are needed. Canine DLBCL, one of the most common cancers in veterinary oncology, is morphologically similar to hDLBCL and is treated using similar chemotherapeutic protocols. With genomic technologies, it is now possible to molecularly evaluate dogs as a potential large-animal model for hDLBCL. We evaluated canine B-cell lymphomas (cBCL) using immunohistochemistry (IHC) and gene expression profiling. cBCL expression profiles were similar in many ways to hDLBCLs. For instance, a subset had increased expression of NF-κB pathway genes, mirroring human activated B-cell (ABC)-type DLBCL. Furthermore, immunoglobulin heavy chain ongoing mutation status, which is correlated with ABC/germinal center B-cell cell of origin in hDLBCL, separated cBCL into two groups with statistically different progression-free and overall survival times. In contrast with hDLBCL, cBCL rarely expressed BCL6 and MUM1/IRF4 by IHC. Collectively, these studies identify molecular similarities to hDLBCL that introduce pet dogs as a representative model of hDLBCL for future studies, including therapeutic clinical trials.

An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation.

B cell lymphoma and melanoma harbor recurrent mutations in the gene encoding the EZH2 histone methyltransferase (EZH2), but the carcinogenic role of these mutations is unclear. Here we describe a mouse model in which the most common somatic Ezh2 gain-of-function mutation (EZH2Y646F in human; Ezh2Y641F in mouse) is conditionally expressed. Expression of Ezh2Y641F in mouse B cells or melanocytes caused high-penetrance lymphoma or melanoma, respectively. Overexpression of the anti-apoptotic protein Bcl2, but not the oncoprotein Myc, or loss of the tumor suppressor protein p53 (encoded by Trp53 in mice) further accelerated lymphoma progression. Expression of the mutant Braf but not the mutant Nras oncoprotein further accelerated melanoma progression. Although expression of Ezh2Y641F globally increased the abundance of trimethylated Lys27 of histone H3 (H3K27me3), it also caused a widespread redistribution of this repressive mark, including a loss of H3K27me3 that was associated with increased transcription at many loci. These results suggest that Ezh2Y641F induces lymphoma and melanoma through a vast reorganization of chromatin structure, inducing both repression and activation of polycomb-regulated loci.B-cell lymphoma and melanoma harbor recurrent mutations in the gene encoding the EZH2 histone methyltransferase, but the carcinogenic role of these mutations is unclear. Here we describe a mouse model in which the most common somatic EZH2 gain-of-function mutation (Y646F in human, Y641F in the mouse) can be conditionally expressed. Expression of Ezh2Y641F in mouse B-cells or melanocytes caused high-penetrance lymphoma or melanoma, respectively. Bcl2 overexpression or p53 loss, but not c-Myc overexpression, further accelerated lymphoma progression, and expression of mutant B-Raf but not mutant N-Ras further accelerated melanoma progression. Although expression of Ezh2Y641F increased abundance of global H3K27 trimethylation (H3K27me3), it also caused a widespread redistribution of this repressive mark, including a loss of H3K27me3 associated with increased transcription at many loci. These results suggest that Ezh2Y641F induces lymphoma and melanoma through a vast reorganization of chromatin structure inducing both repression and activation of polycomb-regulated loci.

Genetic tests to evaluate prognosis and predict therapeutic response in acute myeloid leukemia

Management of patients with acute myeloid leukemia relies on genetic tests that inform diagnosis and prognosis, predict response to therapy, and measure minimal residual disease. The value of genetics is reinforced in the revised 2008 World Health Organization acute myeloid leukemia classification scheme. The various analytic procedures-karyotype, fluorescence in situ hybridization, reverse transcription polymerase chain reaction, DNA sequencing, and microarray technology-each have advantages in certain clinical settings, and understanding their relative merits assists in specimen allocation and in effective utilization of health care resources. Karyotype and array technology represent genome-wide screens, whereas the other methods target specific prognostic features such as t(15;17) PML-RARA, t(8;21) RUNX1-RUNX1T1, inv(16) CBFB-MYH11, 11q23 MLL rearrangement, FLT3 internal tandem duplication, or NPM1 mutation. New biomarkers and pharmacogenetic tests are emerging. The pathologists expertise is critical in 1) consulting with clinicians about test selection as well as specimen collection and handling; 2) allocating tissue for immediate testing and preserving the remaining specimen for any downstream testing that is indicated once morphology and other pertinent test results are known; 3) performing tests that maximize outcome based on the strengths and limitations of each assay in each available specimen type; and 4) interpreting and conveying results to the rest of the health care team in a format that facilitates clinical management. Acute myeloid leukemia leads the way for modern molecular medicine.

Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Expression of six drug transporters in vaginal, cervical, and colorectal tissues: Implications for drug disposition in HIV prevention.

Effective antiretroviral (ARV)‐based HIV prevention strategies require optimizing drug exposure in mucosal tissues; yet factors influencing mucosal tissue disposition remain unknown. We hypothesized drug transporter expression in vaginal, cervical, and colorectal tissues is a contributing factor and selected 3 efflux (ABCB1/MDR1, ABCC2/MRP2, ABCC4/MRP4) and 3 uptake (SLC22A6/OAT1, SLC22A8/OAT3, SLCO1B1/OATP1B1) transporters to further investigate based on their affinity for 2 ARVs central to prevention (tenofovir, maraviroc). Tissue was collected from 98 donors. mRNA and protein expression were quantified using qPCR and immunohistochemistry (IHC). Hundred percent of tissues expressed efflux transporter mRNA. IHC localized them to the epithelium and/or submucosa. Multivariable analysis adjusted for age, smoking, and co‐medications revealed significant (P < 0.05) differences in efflux transporter mRNA between tissue types (vaginal ABCB1 3.9‐fold > colorectal; vaginal ABCC2 2.9‐fold > colorectal; colorectal ABCC4 2.0‐fold > cervical). In contrast, uptake transporter mRNA was expressed in <25% of tissues. OAT1 protein was detected in 0% of female genital tissues and in 100% of colorectal tissues, but only in rare epithelial cells. These data support clinical findings of higher maraviroc and tenofovir concentrations in rectal tissue compared to vaginal or cervical tissue after oral dosing. Quantifying mucosal transporter expression and localization can facilitate ARV selection to target these tissues.

Early Experience after Developing a Pathology Laboratory in Malawi, with Emphasis on Cancer Diagnoses

Background Despite increasing cancer burden in Malawi, pathology services are limited. We describe operations during the first 20 months of a new pathology laboratory in Lilongwe, with emphasis on cancer diagnoses. Methods and Findings We performed a cross-sectional study of specimens from the Kamuzu Central Hospital pathology laboratory between July 1, 2011 and February 28, 2013. Patient and specimen characteristics, and final diagnoses are summarized. Diagnoses were categorized as malignant, premalignant, infectious, other pathology, normal or benign, or nondiagnostic. Patient characteristics associated with premalignancy and malignancy were assessed using logistic regression. Of 2772 specimens, 2758 (99%) with a recorded final diagnosis were included, drawn from 2639 unique patients. Mean age was 38 years and 63% were female. Of those with documented HIV status, 51% had unknown status, and 36% with known status were infected. Histologic specimens comprised 91% of cases, and cytologic specimens 9%. Malignant diagnoses were most common overall (n = 861, 31%). Among cancers, cervical cancer was most common (n = 117, 14%), followed by lymphoma (n = 91, 11%), esophageal cancer (n = 86, 10%), sarcoma excluding Kaposi sarcoma (n = 75, 9%), and breast cancer (n = 61, 7%). HIV status was known for 95 (11%) of malignancies, with HIV prevalence ranging from 9% for breast cancer to 81% for cervical cancer. Increasing age was consistently associated with malignancy [bivariable odds ratio 1.24 per decade increase (95% CI 1.19–1.29) among 2685 patients with known age; multivariable odds ratio 1.33 per decade increase (95% CI 1.14–1.56) among 317 patients with known age, gender, and HIV status], while HIV infection and gender were not. Conclusions Despite selection and referral bias inherent in these data, a new pathology laboratory in Lilongwe has created a robust platform for cancer care and research. Strategies to effectively capture clinical information for pathologically confirmed cancers can allow these data to complement population-based registration.

FOXP1 potentiates Wnt/β-catenin signaling in diffuse large B cell lymphoma

A common type of lymphoma overexpressing the transcription factor FOXP1 could be treated with Wnt/β-catenin inhibitors. Targeting Wnt signaling in lymphoma Although several human cancers show increased activity of the Wnt/β-catenin signaling pathway, tumors may lack mutations in components in this pathway that would account for the increase in activity. Using a gain-of-function screen and various cancer cell lines and in vivo models, Walker et al. found that the transcription factor FOXP1 (forkhead box protein P1) enhanced the transcription of Wnt-regulated target genes by binding to and promoting the acetylation of β-catenin. Patients with diffuse large B cell lymphomas overexpressing FOXP1 have a poor prognosis, and diffuse large B cell lymphoma cells with high FOXP1 abundance were sensitive to Wnt inhibitors. Xenografted tumors in mice were smaller when they lacked FOXP1 or when Wnt signaling was blocked. The transcription factor FOXP1 (forkhead box protein P1) is a master regulator of stem and progenitor cell biology. In diffuse large B cell lymphoma (DLBCL), copy number amplifications and chromosomal translocations result in overexpression of FOXP1. Increased abundance of FOXP1 in DLBCL is a predictor of poor prognosis and resistance to therapy. We developed a genome-wide, mass spectrometry–coupled, gain-of-function genetic screen, which revealed that FOXP1 potentiates β-catenin–dependent, Wnt-dependent gene expression. Gain- and loss-of-function studies in cell models and zebrafish confirmed that FOXP1 was a general and conserved enhancer of Wnt signaling. In a Wnt-dependent fashion, FOXP1 formed a complex with β-catenin, TCF7L2 (transcription factor 7-like 2), and the acetyltransferase CBP [CREB (adenosine 3′,5′-monophosphate response element–binding protein)–binding protein], and this complex bound the promoters of Wnt target genes. FOXP1 promoted the acetylation of β-catenin by CBP, and acetylation was required for FOXP1-mediated potentiation of β-catenin–dependent transcription. In DLBCL, we found that FOXP1 promoted sensitivity to Wnt pathway inhibitors, and knockdown of FOXP1 or blocking β-catenin transcriptional activity slowed xenograft tumor growth. These data connect excessive FOXP1 with β-catenin–dependent signal transduction and provide a molecular rationale for Wnt-directed therapy in DLBCL.

Mass Spectrometry Imaging Reveals Heterogeneous Efavirenz Distribution within Putative HIV Reservoirs

ABSTRACT Persistent HIV replication within active viral reservoirs may be caused by inadequate antiretroviral penetration. Here, we used mass spectrometry imaging with infrared matrix-assisted laser desorption–electrospray ionization to quantify the distribution of efavirenz within tissues from a macaque dosed orally to a steady state. Intratissue efavirenz distribution was heterogeneous, with the drug concentrating in the lamina propria of the colon, the primary follicles of lymph nodes, and the brain gray matter. These are the first imaging data of an antiretroviral drug in active viral reservoirs.