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TO THE EDITOR: Indolent T-cell lymphoproliferative disorder of the gastrointestinal tract (GI TLPD) is a newly recognized entity in the World Health Organization (WHO) classification of lymphoid neoplasms.[1][1] GI TLPD is defined as a clonal T-cell proliferation occurring in the GI tract, most

Targetable fusions of the FRK tyrosine kinase in ALK-negative anaplastic large cell lymphoma

Anaplastic large cell lymphomas (ALCLs) are CD30-positive T-cell non-Hodgkin lymphomas that bear chromosomal rearrangements of the TP53 homologue TP63 in a subset of cases that demonstrate aggressive clinical behavior. In the present study, we examined the relationship between p63 protein expression by immunohistochemistry and the results of fluorescence in situ hybridization using TP63 probes in 116 ALCLs. We also determined the relative expression of full-length TAp63 and truncated ΔNp63 isoforms (eg, p40) in ALCL cell lines and a subset of clinical cases. Overall, 35.3% of ALCLs were positive for p63 protein. Primary cutaneous and anaplastic lymphoma kinase-negative ALCLs were positive more frequently than anaplastic lymphoma kinase-positive ALCLs (P=.0034). As previously reported, cases with TP63 gene rearrangements expressed p63 uniformly. p63 expression in nonrearranged cases was associated with extra copies of TP63 on 3q28 (P

// Xueju Wang 1, 2 , Surendra Dasari 3 , Grzegorz S. Nowakowski 4 , Konstantinos N. Lazaridis 5, 6 , Eric D. Wieben 5, 7 , Marshall E. Kadin 8 , Andrew L. Feldman 1, * , Rebecca L. Boddicker 1, * 1 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, United States of America 2 Department of Pathology, China-Japan Union Hospital of Jilin Province, Changchun, Jilin Province, China 3 Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, United States of America 4 Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America 5 Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota, United States of America 6 Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America 7 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States of America 8 Department of Pathology and Laboratory Medicine, Rhode Island Hospital and Department of Dermatology, Roger Williams Medical Center, Providence, Rhode Island, United States of America * These authors have contributed equally to this work Correspondence to: Andrew L. Feldman, email: feldman.andrew@mayo.edu Rebecca L. Boddicker, email: boddicker.rebecca@mayo.edu Keywords: T-cell lymphoma, retinoids, retinoic acid receptor alpha, all-trans retinoic acid, cell cycle, individualized medicine Received: October 04, 2016 Accepted: February 06, 2017 Published: February 17, 2017 ABSTRACT Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with generally poor outcomes following standard therapy. Few candidate therapeutic targets have been identified to date. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to retinoids. While retinoids have been used to treat some cutaneous T-cell lymphomas (CTCLs), their mechanism of action and the role of RARA in CTCL and other mature T-cell lymphomas remain poorly understood. After identifying a PTCL with a RARA R394Q mutation, we sought to characterize the role of RARA in T-cell lymphoma cells. Overexpressing wild-type RARA or RARA R394Q significantly increased cell growth in RARA low cell lines, while RARA knockdown induced G1 arrest and decreased expression of cyclin-dependent kinases CDK2/4/6 in RARA high cells. The retinoids, AM80 (tamibarotene) and all- trans retinoic acid, caused dose-dependent growth inhibition, G 1 arrest, and CDK2/4/6 down-regulation. Genes down-regulated in transcriptome data were enriched for cell cycle and G1-S transition. Finally, RARA overexpression augmented chemosensitivity to retinoids. In conclusion, RARA drives cyclin-dependent kinase expression, G 1 -S transition, and cell growth in T-cell lymphoma. Synthetic retinoids inhibit these functions in a dose-dependent fashion and are most effective in cells with high RARA expression, indicating RARA may represent a therapeutic target in some PTCLs.

Peripheral T-cell lymphomas (PTCLs) are rare, heterogeneous tumors with poor response to standard therapy and few targeted treatments available. The identification of mutations in the T-cell receptor (TCR) signaling pathway that either directly or indirectly affect Ras- and Rho-family GTPases is an emerging theme across PTCL subtypes. This review summarizes the role of GTPases in TCR signaling and highlights the constellation of mutations in this pathway among PTCLs. In particular, focus is given to the functional impact of the mutations and opportunities for targeted therapy. These mutations include activating mutations and gene fusions involving the guanine nucleotide exchange factor, VAV1, as well as activating and dominant negative mutations in the GTPases KRAS and RHOA, respectively. In addition to mutations directly affecting the GTPase pathway, TCR signaling mutations indirectly affecting Ras- and Rho-family GTPases involving genes such as CD28, FYN, LCK, and PLCG1 are also reviewed.

Classification of anaplastic large cell lymphoma: historical background Anaplastic large cell lymphoma (ALCL) represents a group of non-Hodgkin lymphomas of mature T-cell origin (peripheral T-cell lymphomas [PTCLs]) that share common pathologic features, especially the presence of large lymphocytes that often involve lymph node sinuses and express the Ki-1 antigen, now designated CD30 [1]. A chromosomal rearrangement in a subset of these cases, t(2;5)(p23;q35), was found to involve the nucleophosmin gene, NPM1, and a novel tyrosine kinase gene, designated anaplastic lymphoma kinase or ALK [2]. Immunohistochemical staining for ALK allowed recognition that cases with several histologic patterns as well as cases with variant, nonNPM1 partner genes could be unified into a common clinicopathologic entity, ALK-positive ALCL [3]. An additional group of cases lacked ALK rearrangements, and these ALKnegative ALCLs occurred in older patients and had inferior outcomes compared with ALK-positive ALCLs [4]. The fourth edition of the WHO classification of lymphomas provisionally classified ALK-negative ALCL as a distinct entity separate from ALK-positive ALCL [5], and this provisional status is likely to be dropped in a forthcoming WHO update. Nevertheless, diagnostic criteria for ALK-negative ALCL still are evolving, particularly to distinguish it from other CD30positive PTCLs [5–7]. Primary cutaneous ALCL is recognized separately by the WHO and represents ALCLs that present in the skin and typically have excellent long-term outcomes [5]; these tumors share some genetic features with systemic ALCLs, as discussed below. The pathogenic role of ALK fusions in ALK-positive ALCL has been studied extensively; however, the pathobiology of ALKnegative ALCL remains incompletely understood [6]. Gene expression profiling studies have identified a molecular signature for ALK-positive ALCL and another signature common to all ALCLs [7]. This latter signature can help distinguish ALCL from other PTCLs, but identifying a signature specific for ALK-negative ALCL has proved challenging. This difficulty likely relates in part to intrinsic molecular heterogeneity within the category of ALK-negative ALCL.

Peripheral T-cell lymphomas (PTCLs) are generally aggressive non-Hodgkin lymphomas with poor overall survival rates following standard therapy. One-third of PTCLs express interferon regulatory factor-4 (IRF4), a tightly regulated transcription factor involved in lymphocyte growth and differentiation. IRF4 drives tumor growth in several lymphoid malignancies and has been proposed as a candidate therapeutic target. Because direct IRF4 inhibitors are not clinically available, we sought to characterize the mechanism by which IRF4 expression is regulated in PTCLs. We demonstrated that IRF4 is constitutively expressed in PTCL cells and drives Myc expression and proliferation. Using an inhibitor screen, we identified nuclear factor κB (NF-κB) as a candidate regulator of IRF4 expression and cell proliferation. We then demonstrated that the NF-κB subunits p52 and RelB were transcriptional activators of IRF4. Further analysis showed that activation of CD30 promotes p52 and RelB activity and subsequent IRF4 expression. Finally, we showed that IRF4 transcriptionally regulates CD30 expression. Taken together, these data demonstrate a novel positive feedback loop involving CD30, NF-κB, and IRF4; further evidence for this mechanism was demonstrated in human PTCL tissue samples. Accordingly, NF-κB inhibitors may represent a clinical means to disrupt this feedback loop in IRF4-positive PTCLs.

Peripheral T cell lymphomas (PTCLs) are markedly heterogeneous at the clinical, pathological, and molecular levels. This review will discuss genetic findings in PTCL with special emphasis on how they impact lymphoma classification.Sequencing studies have identified recurrent genetic alterations in nearly every PTCL subtype. In anaplastic large cell lymphoma, these studies have revealed novel chromosomal rearrangements and mutations that have prognostic significance and may suggest new therapeutic approaches. Angioimmunoblastic T cell lymphoma has been found to have mutations overlapping some cases of PTCL, not otherwise specified with a T follicular helper cell phenotype. Across various subtypes, recurrent mutations and structural alterations affecting genes involved in epigenetic regulation, T cell receptor signaling, and immune response may represent targets for precision therapy approaches. New genetic findings are refining the classification of PTCLs and are beginning to be used clinically for diagnosis, risk stratification, and individualized therapy.

In utero hyperthermia can cause a variety of developmental issues, but how it alters mammalian body temperature during adolescence is not well-understood. Study objectives were to determine the extent to which in utero hyperthermia affects future phenotypic responses to a heat load. Pregnant first parity pigs were exposed to thermal neutral (TN) or heat stress (HS) conditions during the entire gestation. Of the resultant offspring, 12 were housed in TN conditions, and 12 were maintained in HS conditions for 15 days. Adolescent pigs in HS conditions had increased rectal temperature and respiration rate (RR) compared to TN pigs, regardless of gestational treatment. Within the HS environment, no gestational difference in RR was detected; however, GHS pigs had increased rectal temperature compared to GTN pigs. As rectal temperature increased, GTN pigs had a more rapid increase in RR compared to the GHS pigs. Adolescent HS decreased nutrient intake, and body weight gain, but neither variable was statistically influenced by gestational treatments. In summary, in utero HS compromises the future thermoregulatory response to a thermal insult.

Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a “wastebasket” category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

Obesity is associated with a decrease in the antiinflammatory hormone, adiponectin, and increases in the circulating concentrations of multiple proinflammatory cytokines. These changes contribute to colon tumorigenesis. Resveratrol increases adiponectin production in adipocytes and attenuates the development of colon cancer. Thus, we hypothesized that adiponectin is an integral component of the mechanism by which resveratrol antagonizes colorectal tumorigenesis. To investigate this, we induced tumorigenesis in adiponectin knockout (KO) and wild-type (Wt) C57BL/6 mice through combined azoxymethane and dextran sodium sulfate treatment during which mice were fed a high-fat, lard-based diet, or the same diet containing 20 mg/kg resveratrol. After 14 wk on diet, Wt mice gained more weight and, on a percentage basis, had higher fat mass and lower lean mass than KO mice. Resveratrol tended to attenuate this response in male Wt mice. Resveratrol also tended to reduce aberrant crypt foci development and decrease circulating interleukin 6 and insulin concentrations in male but not female Wt mice. Taken together, resveratrol improved overall health of obese Wt but not KO mice as hypothesized with a differential sex response.

Background: Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with marked clinical, pathological, and molecular heterogeneity. Outcomes following standard therapy generally are poor; however, few candidate therapeutic targets have been identified for precision medicine approaches. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to natural or synthetic retinoids. Retinoids have been used successfully to treat acute promyelocytic leukemia and some cutaneous T-cell lymphomas (CTCLs). However, the function of RARA and the action of retinoids in PTCL have not been defined. Methods:Based on identification of a PTCL patient with a non-synonymous point mutation, RARA R394Q, identified in the Mayo Clinic Center for Individualized Medicine, we sought to characterize the role of RARA in PTCL cells. To investigate the role of wild-type and mutant RARA, we constructed expression vectors containing either wild-type RARA or RARA R394Q coding sequences, and also used siRNAs targeting RARA to study the role of native RARA expression. Cell lines derived from post-thymic T-cell malignancies were used for in vitro studies, including HuT78 and Mac-1 (both derived from circulating tumor cells from CTCL patients) and Karpas 299 (from an ALK-positive anaplastic large cell lymphoma). Following RARA overexpression or knockdown, we measured cell growth, cell cycle regulation, and sensitivity to synthetic retinoids. In addition, RNA sequencing and pathway analysis were performed to profile the transcriptomic response to retinoids in malignant T cells. Results:In two RARAlow cell lines, Karpas 299 and HuT78, overexpression of wild-type RARA or RARA R394Q significantly increased cell growth (p Conclusions:RARA drives cyclin-dependent kinase expression and G1-S transition in malignant T cells, and promotes cell growth. These functions may be enhanced by specific RARA gene mutations. Synthetic retinoids inhibit these functions in a dose-dependent fashion, and are most effective in cells with high RARA expression. These data suggest RARA as a candidate therapeutic target in some PTCL patients. Disclosures Nowakowski: Celgene: Research Funding; Morphosys: Research Funding; Bayer: Consultancy, Research Funding.

Adiponectin is an antiinflammatory and insulin-sensitizing hormone that is decreased in obesity. Although controversial, it has been suggested that decreased adiponectin contributes to colorectal cancer risk in obesity. To further investigate the role of adiponectin in obesity-linked colorectal carcinogenesis, we used male and female adiponectin knockout (KO) and wild-type (Wt) C57BL/6J mice. Tumorigenesis was induced in all mice with the combined treatment of azoxymethane (AOM) and dextran sodium sulfate (DSS). Following AOM/DSS treatment, mice were fed a low-fat control (LFC), or high-fat lard (HFL) diet for 7 1/2 wk. KO mice developed fewer total lesions than Wt mice, males developed fewer lesions than females, and mice fed the HFL diet developed fewer lesions than those fed the LFC diet. Early lesion multiplicity was influenced by genotype, whereas advanced lesion development was influenced by sex and diet. Moreover, lesion types were differentially correlated with serum adipokines and colon gene expression of adiponectin receptors, insulin receptor, and toll-like receptor 4. These data suggest that in the AOM/DSS model of carcinogenesis, adiponectin functions to promote early lesion development whereas sex and diet are important regulators of advanced lesion development through pathways involved in inflammation and insulin signaling.

Interferon regulatory factor-4 (IRF4) is a tightly-regulated transcription factor involved in growth and differentiation of normal lymphocytes. IRF4 is expressed in multiple lymphoid malignancies and its expression drives tumor growth. Thus, characterization of the molecular events regulating IRF4 levels is key to design any targeting strategy. Here, we define a novel regulatory mechanism controlling IRF4 expression in peripheral T-cell lymphoma (PTCL), an aggressive non-Hodgkin lymphoma with poor overall survival rates and high levels of this transcription factor. In normal T cells, IRF4 was induced upon external activation of protein kinase C (PKC) by phorbol myristate acetate and ionomycin, an effect reversed by the PKC inhibitor, enzastaurin. Unlike normal T cells, PTCL cells constitutively expressed IRF4 in the absence of external stimulation, and this expression was resistant to enzastaurin. To identify regulators of IRF4 expression in PTCL, we performed a pharmacological screen using inhibitors that targeted PKC-associated pathways, including mitogen-activated protein kinases, PI3K/AKT/mTOR, and NF-κB. Of the inhibitors tested, only the proteasome inhibitor, bortezomib, showed a decrease in IRF4 expression in a dose- and time-dependent manner. To confirm this result we used a more specific NF-κB inhibitor blocking IκB kinase, BMS-345541. This inhibitor dramatically reduced IRF4 expression by 95.6% (P Citation Format: Rebecca L. Boddicker, N. Sertac Kip, Luciana L. Almada, Julie C. Porcher, Deanna M. Grote, Stephen M. Ansell, Martin E. Fernandez-Zapico, Andrew L. Feldman. Interferon regulatory factor-4 is transcriptionally regulated by NF-κB in peripheral T-cell lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2323. doi:10.1158/1538-7445.AM2014-2323

Background Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with poor overall survival rates. The genetics of PTCLs are poorly understood. We have described t(6;14)(p25.3;q11.2) translocations in PTCLs involving the T-cell receptor-alpha (TRA@) and interferon regulatory factor-4 (IRF4) genes and overexpressing IRF4 protein (also called MUM1). IRF4/MUM1 is a lymphoid transcription factor involved in differentiation and growth, the expression of which is tightly regulated in normal T cells. However, nearly half of PTCLs constitutively express IRF4/MUM1 and this expression is associated with adverse overall survival in several PTCL subtypes. Because IRF4/TRA@ translocations are rare among PTCLs ( Methods Studies were performed on paraffin tissue sections of PTCL specimens diagnosed by standard WHO criteria. IRF4/MUM1 immunohistochemical staining (IHC) was performed using the MUM1p antibody and considered positive if nuclear staining was present in >30% of tumor cell nuclei. Fluorescence in situ hybridization (FISH) was performed using breakapart probes constructed from bacterial artificial chromosome DNA that hybridized to loci flanking the IRF4 locus on 6p25.3. Cases with 3 or more fusion signals were considered to have extra copies of the IRF4 gene. In a subset of cases, PCR and Sanger sequencing was performed on DNA extracted from frozen PTCL tumor tissue with primer sets that amplified exons 1 to 10 of the IRF4 gene. Data were analyzed using the chi-square test or t test. Results PTCLs from 277 patients (175 M:102 F; median age, 61 y) were examined by both IHC and FISH. The subtype distribution was: PTCL, not otherwise specified (NOS), 93; anaplastic large cell lymphoma (ALCL), 89; angioimmunoblastic T-cell lymphoma (AITL), 27; cutaneous T-cell lymphoma (CTCL), 43; and cytotoxic PTCL, 25. IRF4/MUM1 was positive in 116 cases (42%). Extra copies of IRF4 were identified in 18 PTCLs (7%), 83% of which were IRF4/MUM1 protein-positive. In contrast, 39% of cases without extra copies of IRF4 were IRF4/MUM1 protein-positive (p=0.0002). The mean percentage of tumor cells with IRF4/MUM1 staining was 69% in cases with extra copies of IRF4, compared to 30% in cases without extra copies (p=0.0001). Extra copies of IRF4 were seen in 13% of ALCLs, 4% each of PTCLs, NOS and cytotoxic PTCLs, 2% of CTCLs, and 0% of AITLs (p=0.02). Two IRF4/MUM1-positive cases had an IRF4/TRA@ translocation (1%). We selected 30 IRF4/MUM1-positive PTCLs for Sanger sequencing. In this subset, we identified a previously undescribed non-synonymous variant in exon 5 (S309R) in 1 case (3%) without an extra copy of IRF4. Conclusions Extra copies of IRF4 are seen in ∼7% of PTCLs, are significantly associated with IRF4/MUM1 expression, and are preferentially observed in ALCLs. Both IRF4/TRA@ translocations and IRF4 mutations are rare in PTCLs. These data suggest that genetic abnormalities of the IRF4 gene – specifically extra copies of IRF4 – may contribute to IRF4/MUM1 expression in some PTCLs. Since IRF4/MUM1 expression is seen in ∼42% of PTCLs, these data also imply the existence of other regulatory mechanisms. Characterizing these mechanisms may be important clinically, since IRF4/MUM1 has been proposed as a therapeutic target in human cancers but drugs that directly inhibit its function are currently not available. Disclosures: No relevant conflicts of interest to declare.