Adult high-grade B-cell lymphoma with Burkitt lymphoma signature: genomic features and potential therapeutic targets.

The adult high-grade B-cell lymphomas sharing molecular features with Burkitt lymphoma (BL) are highly aggressive lymphomas with poor clinical outcome. High-resolution structural and functional genomic analysis of adult Burkitt lymphoma (BL) and high-grade B-cell lymphoma with BL gene signature (adult-molecularly defined BL [mBL]) revealed the MYC-ARF-p53 axis as the primary deregulated pathway. Adult-mBL had either unique or more frequent genomic aberrations (del13q14, del17p, gain8q24, and gain18q21) compared with pediatric-mBL, but shared commonly mutated genes. Mutations in genes promoting the tonic B-cell receptor (BCR)→PI3K pathway (TCF3 and ID3) did not differ by age, whereas effectors of chronic BCR→NF-κB signaling were associated with adult-mBL. A subset of adult-mBL had BCL2 translocation and mutation and elevated BCL2 mRNA and protein expression, but had a mutation profile similar to mBL. These double-hit lymphomas may have arisen from a tumor precursor that acquired both BCL2 and MYC translocations and/or KMT2D (MLL2) mutation. Gain/amplification of MIR17HG and its paralogue loci was observed in 50% of adult-mBL. In vitro studies suggested miR-17∼92's role in constitutive activation of BCR signaling and sensitivity to ibrutinib. Overall integrative analysis identified an interrelated gene network affected by copy number and mutation, leading to disruption of the p53 pathway and the BCR→PI3K or NF-κB activation, which can be further exploited in vivo by small-molecule inhibitors for effective therapy in adult-mBL.

Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma.

We studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)-DLBCL compared with activated B-cell (ABC)-DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the "gold standard" GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL.

MicroRNA expression profiling identifies molecular signatures associated with anaplastic large cell lymphoma.

Anaplastic large-cell lymphomas (ALCLs) encompass at least 2 systemic diseases distinguished by the presence or absence of anaplastic lymphoma kinase (ALK) expression. We performed genome-wide microRNA (miRNA) profiling on 33 ALK-positive (ALK[+]) ALCLs, 25 ALK-negative (ALK[-]) ALCLs, 9 angioimmunoblastic T-cell lymphomas, 11 peripheral T-cell lymphomas not otherwise specified (PTCLNOS), and normal T cells, and demonstrated that ALCLs express many of the miRNAs that are highly expressed in normal T cells with the prominent exception of miR-146a. Unsupervised hierarchical clustering demonstrated distinct clustering of ALCL, PTCL-NOS, and the AITL subtype of PTCL. Cases of ALK(+) ALCL and ALK(-) ALCL were interspersed in unsupervised analysis, suggesting a close relationship at the molecular level. We identified an miRNA signature of 7 miRNAs (5 upregulated: miR-512-3p, miR-886-5p, miR-886-3p, miR-708, miR-135b; 2 downregulated: miR-146a, miR-155) significantly associated with ALK(+) ALCL cases. In addition, we derived an 11-miRNA signature (4 upregulated: miR-210, miR-197, miR-191, miR-512-3p; 7 downregulated: miR-451, miR-146a, miR-22, miR-455-3p, miR-455-5p, miR-143, miR-494) that differentiates ALK(-) ALCL from other PTCLs. Our in vitro studies identified a set of 32 miRNAs associated with ALK expression. Of these, the miR-17∼92 cluster and its paralogues were also highly expressed in ALK(+) ALCL and may represent important downstream effectors of the ALK oncogenic pathway.

Genome-wide miRNA profiling of mantle cell lymphoma reveals a distinct subgroup with poor prognosis.

miRNA deregulation has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Using a high-throughput quantitative real-time PCR platform, we performed miRNA profiling on cyclin D1-positive MCL (n = 30) and cyclin D1-negative MCL (n = 7) and compared them with small lymphocytic leukemia/lymphoma (n = 12), aggressive B-cell lymphomas (n = 138), normal B-cell subsets, and stromal cells. We identified a 19-miRNA classifier that included 6 up-regulated miRNAs and 13 down regulated miRNA that was able to distinguish MCL from other aggressive lymphomas. Some of the up-regulated miRNAs are highly expressed in naive B cells. This miRNA classifier showed consistent results in formalin-fixed paraffin-embedded tissues and was able to distinguish cyclin D1-negative MCL from other lymphomas. A 26-miRNA classifier could distinguish MCL from small lymphocytic leukemia/lymphoma, dominated by 23 up-regulated miRNAs in MCL. Unsupervised hierarchical clustering of MCL patients demonstrated a cluster characterized by high expression of miRNAs from the polycistronic miR17-92 cluster and its paralogs, miR-106a-363 and miR-106b-25, and associated with high proliferation gene signature. The other clusters showed enrichment of stroma-associated miRNAs, and also had higher expression of stroma-associated genes. Our clinical outcome analysis in the present study suggested that miRNAs can serve as prognosticators.

Genetic bases of estrogen-induced tumorigenesis in the rat: mapping of loci controlling susceptibility to mammary cancer in a Brown Norway x ACI intercross.

Exposure to estrogens is associated with an increased risk of breast cancer. Our laboratory has shown that the ACI rat is uniquely susceptible to 17beta-estradiol (E2)-induced mammary cancer. We previously mapped two loci, Emca1 and Emca2 (estrogen-induced mammary cancer), that act independently to determine susceptibility to E2-induced mammary cancer in crosses between the susceptible ACI rat strain and the genetically related, but resistant, Copenhagen (COP) rat strain. In this study, we evaluate susceptibility to E2-induced mammary cancer in a cross between the ACI strain and the unrelated Brown Norway (BN) rat strain. Whereas nearly 100% of the ACI rats developed mammary cancer when treated continuously with E2, BN rats did not develop palpable mammary cancer during the 196-day course of E2 treatment. Susceptibility to E2-induced mammary cancer segregated as a dominant or incompletely dominant trait in a cross between BN females and ACI males. In a population of 251 female (BN x ACI)F(2) rats, we observed evidence for a total of five genetic determinants of susceptibility. Two loci, Emca4 and Emca5, were identified when mammary cancer status at sacrifice was evaluated as the phenotype, and three additional loci, Emca6, Emca7, and Emca8, were identified when mammary cancer number was evaluated as the phenotype. A total of three genetic interactions were identified. These data indicate that susceptibility to E2-induced mammary cancer in the BN x ACI cross behaves as a complex trait controlled by at least five loci and multiple gene-gene interactions.

Genetic determination of susceptibility to estrogen-induced mammary cancer in the ACI rat: mapping of Emca1 and Emca2 to chromosomes 5 and 18.

Hormonal, genetic, and environmental factors play major roles in the complex etiology of breast cancer. When treated continuously with 17beta-estradiol (E2), the ACI rat exhibits a genetically conferred propensity to develop mammary cancer. The susceptibility of the ACI rat to E2-induced mammary cancer appears to segregate as an incompletely dominant trait in crosses to the resistant Copenhagen (COP) strain. In both (ACI x COP)F(2) and (COP x ACI)F(2) populations, we find strong evidence for a major genetic determinant of susceptibility to E2-induced mammary cancer on distal rat chromosome 5. Our data are most consistent with a model in which the ACI allele of this locus, termed Emca1 (estrogen-induced mammary cancer 1), acts in an incompletely dominant manner to increase both tumor incidence and tumor multiplicity as well as to reduce tumor latency in these populations. We also find evidence suggestive of a second locus, Emca2, on chromosome 18 in the (ACI x COP)F(2) population. The ACI allele of Emca2 acts in a dominant manner to increase incidence and decrease latency. Together, Emca1 and Emca2 act independently to modify susceptibility to E2-induced mammary cancer.

Genetic etiology of renal agenesis: fine mapping of Renag1 and identification of Kit as the candidate functional gene.

Congenital anomalies of the kidney and urogenital tract (CAKUT) occur in approximately 0.5% of live births and represent the most frequent cause of end-stage renal disease in neonates and children. The genetic basis of CAKUT is not well defined. To understand more fully the genetic basis of one type of CAKUT, unilateral renal agenesis (URA), we are studying inbred ACI rats, which spontaneously exhibit URA and associated urogenital anomalies at an incidence of approximately 10%. URA is inherited as an incompletely dominant trait with incomplete penetrance in crosses between ACI and Brown Norway (BN) rats and a single responsible genetic locus, designated Renag1, was previously mapped to rat chromosome 14 (RNO14). The goals of this study were to fine map Renag1, identify the causal genetic variant responsible for URA, confirm that the Renag1 variant is the sole determinant of URA in the ACI rat, and define the embryologic basis of URA in this rat model. Data presented herein localize Renag1 to a 379 kilobase (kb) interval that contains a single protein coding gene, Kit (v-kit Hardy-Zukerman 4 feline sarcoma viral oncogene homolog); identify an endogenous retrovirus-derived long terminal repeat located within Kit intron 1 as the probable causal variant; demonstrate aberrant development of the nephric duct in the anticipated number of ACI rat embryos; and demonstrate expression of Kit and Kit ligand (Kitlg) in the nephric duct. Congenic rats that harbor ACI alleles at Renag1 on the BN genetic background exhibit the same spectrum of urogenital anomalies as ACI rats, indicating that Renag1 is necessary and sufficient to elicit URA and associated urogenital anomalies. These data reveal the first genetic link between Kit and URA and illustrate the value of the ACI rat as a model for defining the mechanisms and cell types in which Kit functions during urogenital development.

Genetic control of estrogen action in the rat: mapping of QTLs that impact pituitary lactotroph hyperplasia in a BN x ACI intercross.

Estrogens are important regulators of growth and development and contribute to the etiology of several types of cancer. Different inbred rat strains exhibit marked, cell-type-specific differences in responsiveness to estrogens as well as differences in susceptibility to estrogen-induced tumorigenesis. Regulation of pituitary lactotroph homeostasis is one estrogen-regulated response that differs dramatically between different inbred rat strains. In this article we demonstrate that the growth response of the anterior pituitary gland of female ACI rats to 17beta-estradiol (E2) markedly exceeds that of identically treated female Brown Norway (BN) rats. We further demonstrate that pituitary mass, a surrogate indicator of absolute lactotroph number, behaves as a quantitative trait in E2-treated F(2) progeny generated in a genetic cross originating with BN females and ACI males. Composite interval mapping analyses of the (BNxACI)F(2) population revealed quantitative trait loci (QTLs) that exert significant effects on E2-induced pituitary growth on rat chromosome 4 (RNO4) (Ept5) and RNO7 (Ept7). Continuous treatment with E2 rapidly induces mammary cancer in female ACI rats but not BN rats, and QTLs that impact susceptibility to E2-induced mammary cancer in the (BNxACI)F(2) population described here have been mapped to RNO3 (Emca5), RNO4 (Emca6), RNO5 (Emca8), RNO6 (Emca7), and RNO7 (Emca4). Ept5 and Emca6 map to distinct regions of RNO4. However, Ept7 and Emca4 map to the same region of RNO7. No correlation between pituitary mass and mammary cancer number at necropsy was observed within the (BNxACI)F(2) population. This observation, together with the QTL mapping data, indicate that with the exception of the Ept7/Emca4 locus on RNO7, the genetic determinants of E2-induced pituitary growth differ from the genetic determinants of susceptibility to E2-induced mammary cancer.

Genetic bases of renal agenesis in the ACI rat: mapping of Renag1 to chromosome 14.

Unilateral renal agenesis (URA) is a common developmental defect in humans, occurring at a frequency of approximately 1 in 500-1,000 births. Several genetic syndromes include bilateral or unilateral renal agenesis as an associated phenotype. However, URA frequently occurs in individuals not afflicted by these syndromes and is often asymptomatic. Although it is clear that genetic factors contribute to the etiology of URA, the genetic bases of URA are poorly defined at this time. ACI rats, both males and females, exhibit URA at an incidence of 5%-15%. In this article we characterize the incidence of URA in female and male F(1), F(2), and backcross (BC) progeny from reciprocal genetic crosses between the ACI strain and the unaffected Brown Norway (BN) strain. Through interval mapping analyses of 353 phenotypically defined female F(2) progeny, we mapped to rat Chromosome 14 (RNO14) a genetic locus, designated Renag1 (Renal agenesis 1), that serves as the major determinant of URA in these crosses. Further genotypic analyses of URA-affected female and male F(2) and BC progeny localized Renag1 to a 14.4-Mb interval on RNO14 bounded by markers D14Rat50 and D14Rat12. The data from these genetic studies suggest that the ACI allele of Renag1 acts in an incompletely dominant and incompletely penetrant manner to confer URA.

Genetic mapping of Eutr1, a locus controlling E2-induced pyometritis in the Brown Norway rat, to RNO5.

In certain rat strains, chronic estrogen administration can lead to pyometritis, an inflammation of the uterus accompanied by infection and the accumulation of intraluminal pus. In this article, we report that the Brown Norway (BN) rat is highly susceptible to pyometritis induced by 17beta-estradiol (E2). The susceptibility of the BN rat to E2-induced pyometritis appears to segregate as a recessive trait in crosses to the resistant August x Copenhagen Irish (ACI) strain. In a (BN x ACI)F(2) population, we find strong evidence for a major genetic determinant of susceptibility to E2-induced pyometritis on rat chromosome 5 (RNO5). Our data are most consistent with a model in which the BN allele of this locus, designated Eutr1 (Estrogen-induced uterine response 1), acts in an incompletely dominant manner to control E2-induced pyometritis. Furthermore, we have confirmed the contribution of Eutr1 to E2-induced uterine pyometritis using an RNO5 congenic rat strain. In addition to Eutr1, we obtained evidence suggestive of linkage for five additional loci on RNO2, 4, 11, 17, and X that control susceptibility to E2-induced pyometritis in the (BN x ACI)F(2) population.