A phase 1 trial of Vorinostat in combination with concurrent chemoradiation therapy in the treatment of advanced staged head and neck squamous cell carcinoma.

Purpose Vorinostat is a potent HDAC inhibitor that sensitizes head and neck squamous cell carcinoma (HNSCC) to cytotoxic therapy while sparing normal epithelium. The primary objective of this Phase I study was to determine the maximally tolerated dose (MTD) and safety of Vorinostat in combination with standard chemoradiation therapy treatment in HNSCC. Patients and Methods Eligible patients had pathologically confirmed Stage III, IVa, IVb HNSCC, that was unresectable or borderline resectable involving the larynx, hypopharynx, nasopharynx, and oropharynx. Vorinostat was administered at the assigned dosage level (100-400 mg, three times weekly) in a standard 3 + 3 dose escalation design. Vorinostat therapy began 1 week prior to initiation of standard, concurrent chemoradiation therapy and continued during the entire course of therapy. Results Twenty six patients met eligibility criteria and completed the entire protocol. The primary tumor sites included tonsil (12), base of tongue (9), posterior pharyngeal wall (1), larynx (4) and hypopharynx (3). Of the 26 patients, 17 were HPV-positive and 9 were HPV-negative. The MTD of Vorinostat was 300 mg administered every other day. Anemia (n = 23/26) and leukopenia (n = 20/26) were the most commonly identified toxicities. The most common Grade3/4 events included leukopenia (n = 11) and lymphopenia (n = 17). No patient had Grade IV mucositis, dermatitis or xerostomia. The median follow time was 33.8 months (range 1.6-82.9 months). Twenty four of 26 (96.2%) patients had a complete response to therapy. Conclusion Vorinostat in combination with concurrent chemoradiation therapy is a safe and highly effective treatment regimen in HNSCC. There was a high rate of complete response to therapy with toxicity rates comparable, if not favorable to existing therapies. Further investigation in Phase II and III trials is strongly recommended.

Alemtuzumab (Campath-1H) in the treatment of chronic lymphocytic leukemia.

Alemtuzumab (Campath-1H) is a humanized IgG1 monoclonal antibody that targets the human CD52 antigen. CD52 is expressed by a variety of lymphoid neoplasms and most human mononuclear cell subsets. In 2001, alemtuzumab was approved for marketing in the United States and Europe for use in patients with fludarabine-refractory chronic lymphocytic leukemia (CLL). In heavily pretreated patients with CLL, the overall response rate (ORR) is approximately 35%, and in previously untreated patients the ORR is greater than 80%, with a recent randomized study suggesting it is superior to alkylator-based therapy. Importantly, alemtuzumab is effective in patients with high-risk del(17p13.1) and del(11q22.3) CLL. Alemtuzumab combination studies with fludarabine and/or monoclonal antibodies such as rituximab have demonstrated promising results. Alemtuzumab is also being studied in CLL patients as consolidation therapy for treatment of minimal residual disease, in preparation for stem cell transplantation and to prevent acute and chronic graft versus host disease. Alemtuzumab is frequently associated with acute 'first-dose' reactions when administered intravenously, but is much better tolerated when administered subcutaneously without loss of therapeutic efficacy. Additional potential adverse events associated with alemtuzumab administration include myelosuppression as well as profound cellular immune dysfunction with the associated risk of viral reactivation and other opportunistic infections. Additional studies detailing the mechanism of action of alemtuzumab as well as new strategies for prevention of opportunistic infections will aid in the future therapeutic development of this agent.

Endogenous production of interleukin 15 by activated human monocytes is critical for optimal production of interferon-gamma by natural killer cells in vitro.

Natural killer (NK) cells are large granular lymphocytes that constitutively express functional IL-2 receptors. We have shown that recombinant human IL-15 uses the IL-2 receptor to activate human NK cells and can synergize with recombinant human IL-12 to stimulate NK cell production of IFN-gamma in vitro. IFN-gamma production by NK cells is critical in the prevention of overwhelming infection by obligate intracellular microbial pathogens in several experimental animal models. Herein, we demonstrate that human monocytes produce IL-15 protein within 5 h of activation with LPS. Using an IL-15-neutralizing antiserum in a coculture of LPS-activated monocytes and NK cells, we demonstrate that monocyte-derived IL-15 is critical for optimal NK cell production of IFN-gamma. Endogenous IL-15 activates NK cells through the IL-2 receptor, and with endogenous IL-12, regulates NK cell IFN-gamma after monocyte activation by LPS. These in vitro studies are the first to characterize a function for endogenous IL-15, and as such, suggest an important role for IL-15 during the innate immune response. IL-15 may be an important ligand for the NK cell IL-2 receptor in vivo.

Ultra low dose interleukin-2 therapy promotes a type 1 cytokine profile in vivo in patients with AIDS and AIDS-associated malignancies.

This study was undertaken to determine if prolonged daily subcutaneous administration of ultra low dose IL-2 could influence the constitutive endogenous production of a type 1 (IFN-gamma) cytokine in patients with AIDS or AIDS-associated malignancies. Using a quantitative reverse transcription PCR assay, we demonstrate that daily administration of one type 1 cytokine, IL-2, for 3 mo increases significantly the constitutive endogenous gene expression of another type 1 cytokine, IFN-gamma, in vivo. The predominant source of IFN-gamma appears to be IL-2-expanded natural killer cells and CD8(+) T cells. Moreover, PBMC obtained from these patients during IL-2 therapy showed normalization of a profound deficit in IFN-gamma protein production after stimulation with extracts from infectious agents in vitro. Our data suggest that prolonged exogenous administration of a type 1 cytokine in a nontoxic fashion to patients with AIDS and AIDS-associated malignancies can enhance significantly the endogenous type 1 cytokine profile in vivo. Consequently, ultra low dose IL-2 therapy has the potential to improve the immunodeficient hosts' immune response to infectious pathogens that require IFN-gamma for clearance.

GM-CSF and IL-2 induce specific cellular immunity and provide protection against Epstein-Barr virus lymphoproliferative disorder.

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is a potentially life-threatening complication in immune-deficient patients. We have used the severe combined immune deficient (SCID) mouse engrafted with human leukocytes (hu-PBL-SCID) to evaluate the use of human cytokines in the prevention of EBV-LPD in vivo. Daily low-dose IL-2 therapy can prevent EBV-LPD in the hu-PBL-SCID mouse, but protection is lost if murine natural killer (NK) cells are depleted. Here we demonstrate that combined therapy with human GM-CSF and low-dose IL-2 is capable of preventing EBV-LPD in the hu-PBL-SCID mouse in the absence of murine NK cells. Lymphocyte depletion experiments showed that human NK cells, CD8(+) T cells, and monocytes were each required for the protective effects of GM-CSF and IL-2 combination therapy. This treatment resulted in a marked expansion of human CD3(+)CD8(+) lymphocytes in vivo. Using HLA tetramers complexed with EBV immunodominant peptides, a subset of these lymphocytes was found to be EBV-specific. These data establish that combined GM-CSF and low-dose IL-2 therapy can prevent the immune deficiencies that lead to fatal EBV-LPD in the hu-PBL-SCID mouse depleted of murine NK cells, and they point to a critical role for several human cellular subsets in mediating this protective effect.

PRMT5-PTEN molecular pathway regulates senescence and self-renewal of primary glioblastoma neurosphere cells.

Glioblastoma (GBM) represents the most common and aggressive histologic subtype among malignant astrocytoma and is associated with poor outcomes because of heterogeneous tumour cell population including mature non-stem-like cell and immature stem-like cells within the tumour. Thus, it is critical to find new target-specific therapeutic modalities. Protein arginine methyltransferase enzyme 5 (PRMT5) regulates many cellular processes through its methylation activity and its overexpression in GBM is associated with more aggressive disease. Previously, we have shown that silencing of PRMT5 expression in differentiated GBM cell lines results in apoptosis and reduced tumour growth in mice. Here, we report the critical role of PRMT5 in GBM differentiated cells (GBMDC) grown in serum and GBM neurospheres (GBMNS) grown as neurospheres in vitro. Our results uncover a very significant role for PRMT5 in GBMNS self-renewal capacity and proliferation. PRMT5 knockdown in GBMDC led to apoptosis, knockdown in GBMNS led to G1 cell cycle arrest through upregulation of p27 and hypophoshorylation of retinoblastoma protein, leading to senescence. Comparison of impact of PRMT5 on cellular signalling by the Human Phospho-Kinase Array and chromatin immunoprecipitation-PCR revealed that unlike GBMDC, PRMT5 regulates PTEN expression and controls Akt and ERk activity in GBMNS. In vivo transient depletion of PRMT5 decreased intracranial tumour size and growth rate in mice implanted with both primary tumour-derived GBMNS and GBMDC. This is the first study to identify PTEN as a potential downstream target of PRMT5 and PRMT5 is vital to support both mature and immature GBM tumour cell populations.

The dual epigenetic role of PRMT5 in acute myeloid leukemia: gene activation and repression via histone arginine methylation.

Changes in the enzymatic activity of protein arginine methyltransferase (PRMT) 5 have been associated with cancer; however, the protein's role in acute myeloid leukemia (AML) has not been fully evaluated. Here, we show that increased PRMT5 activity enhanced AML growth in vitro and in vivo while PRMT5 downregulation reduced it. In AML cells, PRMT5 interacted with Sp1 in a transcription repressor complex and silenced miR-29b preferentially via dimethylation of histone 4 arginine residue H4R3. As Sp1 is also a bona fide target of miR-29b, the miR silencing resulted in increased Sp1. This event in turn led to transcription activation of FLT3, a gene that encodes a receptor tyrosine kinase. Inhibition of PRMT5 via sh/siRNA or a first-in-class small-molecule inhibitor (HLCL-61) resulted in significantly increased expression of miR-29b and consequent suppression of Sp1 and FLT3 in AML cells. As a result, significant antileukemic activity was achieved. Collectively, our data support a novel leukemogenic mechanism in AML where PRMT5 mediates both silencing and transcription of genes that participate in a 'yin-yang' functional network supporting leukemia growth. As FLT3 is often mutated in AML and pharmacologic inhibition of PRMT5 appears feasible, the PRMT5-miR-29b-FLT3 network should be further explored as a novel therapeutic target for AML.

The Epstein-Barr Virus (EBV) in T Cell and NK Cell Lymphomas: Time for a Reassessment.

While Epstein-Barr virus (EBV) was initially discovered and characterized as an oncogenic virus in B cell neoplasms, it also plays a complex and multifaceted role in T/NK cell lymphomas. In B cell lymphomas, EBV-encoded proteins have been shown to directly promote immortalization and proliferation through stimulation of the NF-κB pathway and increased expression of anti-apoptotic genes. In the context of mature T/NK lymphomas (MTNKL), with the possible exception on extranodal NK/T cell lymphoma (ENKTL), the virus likely plays a more diverse and nuanced role. EBV has been shown to shape the tumor microenvironment by promoting Th2-skewed T cell responses and by increasing the expression of the immune checkpoint ligand PD-L1. The type of cell infected, the amount of plasma EBV DNA, and the degree of viral lytic replication have all been proposed to have prognostic value in T/NK cell lymphomas. Latency patterns of EBV infection have been defined using EBV-infected B cell models and have not been definitively established in T/NK cell lymphomas. Identifying the expression profile of EBV lytic proteins could allow for individualized therapy with the use of antiviral medications. More work needs to be done to determine whether EBV-associated MTNKL have distinct biological and clinical features, which can be leveraged for risk stratification, disease monitoring, and therapeutic purposes.

Pemphigus antibodies fixation and keratinocyte differentiation in organ cultures. Effects of some agents influencing the intracellular content of cyclic AMP.

The effect of some agents, influencing the cyclic adenosine 3', 5'-monophosphate (cAMP) content of human cells, on the ability of the keratinocytes of binding pemphigus antibodies was studied by using tissue cultures of rabbit esophagus. As demonstrated by immunofluorescence (IF) for IgG, the bound antibodies appeared markedly decreased on esophagus explants grown under standard conditions, that is without test agents, when compared to ones fixed on fresh esophagus. But the IF reaction was remarkably more intense when methylxanthines or epinephrine were added to the growth medium of the cultures. Following the addition of these agents to the cultures some histologic modifications appeared in the explants, indicating that the keratinization process had probably been stimulated. This temporal relationship of immunofluorescence and histologic findings seems to suggest the hypothesis that keratinocyte differentiation, regulation of cAMP intracellular content, and pemphigus antibodies fixation are related processes.

DNA-repair after UV-irradiation in skin fibroblasts from patients with actinic keratosis.

Autoradiographic counting technique was utilized to measure the ultraviolet-induced unscheduled DNA synthesis of skin fibroblasts from 12 patients with chronic actinic keratosis and from 12 healthy donors of about the same age. In order to reveal a possible regional difference of DNA repair between the parts of the body ordinarily exposed and those parts unexposed to sunlight, two cell strains were used for each examined subject; one developed from the forehead skin and the other from the abdominal or axillary skin. Unscheduled DNA synthesis appeared depressed in actinic keratosis patients, as compared with controls. In all examined subjects however cell strains from exposed skin showed a DNA repair more active than cell strains from unexposed skin. These findings show that skin cancer may be promoted in actinic keratosis patients by a defect of DNA repair. The exalted DNA repair of chronically sun exposed skin is probably the consequence of a defensive process caused by enzymatic induction.

Association of pre-transplantation positron emission tomography/computed tomography and outcome in mantle cell lymphoma.

Positron emission tomography/computed tomography (PET/CT)-positive findings before autologous SCT (auto-SCT) are associated with inferior PFS and OS in patients with relapsed Hodgkin's and diffuse large B-cell lymphoma. We classified pre-transplant PET/CT performed before auto-SCT as positive or negative to evaluate the impact of pre-transplant PET/CT in mantle cell lymphoma (MCL). In 29 patients, 17 were PET/CT(-) and 12 were PET/CT(+). PET/CT(+) patients were younger (P=0.04), had lower MCL International Prognostic Index (MIPI, P=0.04) scores, but increased bulky adenopathy >5 cm (45% vs 13%, P=0.09). With a median follow-up of 27 months (range: 5-55 months), 7 patients relapsed (4 in the PET/CT(-) group and 3 in the PET/CT(+) group) with 2 deaths in the PET/CT(+) group without a documented relapse. The estimated 2-year PFS was 64% (95% confidence interval (CI): 0.30-0.85) vs 87% (95% CI: 0.57-0.97) in PET/CT(+) and PET/CT(-) patients, respectively (P=0.054). OS was significantly decreased in PET/CT(+) patients (P=0.007), with 2-year estimates of 60% (95% CI: 0.23-0.84) vs 100% in PET/CT(-) patients. A positive pre-transplant PET/CT is associated with a poor prognosis in patients with MCL. Additional factors may impact the prognostic value of PET/CT, as several PET/CT(+) patients remain in remission.

Low-dose interleukin 2 prevents the development of Epstein-Barr virus (EBV)-associated lymphoproliferative disease in scid/scid mice reconstituted i.p. with EBV-seropositive human peripheral blood lymphocytes.

When severe combined immune deficient (SCID) mice undergo i.p. injection with peripheral blood lymphocytes from normal human donors seropositive for EBV, a majority of these mice (hu-PBL-SCID mouse model) subsequently develop a fatal EBV+ lymphoproliferative disease (EBV-LPD) of human B-cell origin. Because T cells normally are critical in the control of EBV infection, we hypothesized that human T-cell dysfunction accounts for EBV-LPD in the hu-PBL-SCID mouse and that systemic administration of T-cell-derived cytokines would reestablish protective immunity against EBV-LPD. We show that the daily s.c. administration of a very low dose (500 international units) of polyethylene glycol-modified recombinant human interleukin 2 (PEG-IL-2) to hu-PBL-SCID mice can prevent the development of fatal EBV-LPD and significantly improves survival (78%), compared with the survival of hu-PBL-SCID mice treated with placebo (20%, P = 0.0008). Additional lymphocyte-depletion experiments showed that mouse natural killer cells and human CD8+ T cells provided cellular immunity necessary for the PEG-IL-2-mediated protective effect, whereas i.p. injection of human peripheral blood lymphocytes depleted of CD4+ T cells had no adverse effect when combined with PEG-IL-2 therapy and may have been beneficial. These data establish that very low-dose PEG-IL-2 therapy can overcome the immune deficiencies that lead to EBV-LPD in the hu-PBL-SCID mouse and point to the usefulness of this model for evaluating cytokine therapies in EBV-LPD. The use of low-dose IL-2 as a preventative immune therapy has potential application in immunocompromised individuals at high risk for EBV-LPD.

Cytokines in the evolution and treatment of AIDS-lymphoma.

AIDS-lymphoma is a heterogeneous disease that most likely results from the complex interaction of several contributing factors, including chronic antigenic activation of B lymphocytes, dysregulated cytokine and co-stimulatory networks, infection with potentially oncogenic viruses (human herpesvirus-8 [HHV-8], Epstein-Barr virus), and accumulation of secondary genetic mutations. Cytokines are believed to play an important role in the immunologic decline that favors opportunistic infection and malignancy in advanced HIV infection. Recent work has provided some evidence that cytokine therapy can partially reverse the immune dysregulation seen in AIDS. This suggests that cytokines are likely to have an important role in both the pathogenesis and treatment or prevention of AIDS-lymphoma.

The c-kit ligand suppresses apoptosis of human natural killer cells through the upregulation of bcl-2.

The bcl-2 protein plays a central role in the regulation of programmed cell death in a variety of tissues and is pivotal to the survival of lymphocytes in vivo. The growth factors responsible for survival of normal lymphocytes are unknown but are likely to maintain viability in part through the regulation of bcl-2 expression. A subset of human natural killer (NK) cells (CD3-CD56bright) are unique among lymphocytes in their constitutive expression of c-kit, a tyrosine kinase cell surface receptor that binds c-kit ligand (KL). Alone, KL does not promote proliferation or further differentiation of CD56bright NK cells. We now report that, in the absence of serum or additional growth factors, KL prevents apoptosis of cultured CD56bright NK cells, as assessed by DNA fragmentation studies, and maintains viability, as measured by biologic responses (i.e., proliferation and cytotoxicity) to the subsequent addition of other cytokines. Furthermore, we demonstrate that KL induces CD56bright NK cells to express the bcl-2 protein. In the presence of anti-c-kit antibody, the tyrosine kinase inhibitor genistein, or bcl-2 antisense oligonucleotide, the protective effect of KL on the survival of CD56bright NK cells is dramatically reduced. These data demonstrate that the binding of KL to its tyrosine kinase receptor results in the upregulation of bcl-2, thereby preventing apoptosis in this subset of normal human lymphocytes. As soluble KL is plentiful in normal human serum, this survival mechanism may be operative for CD56bright NK cells in vivo.

Immunotherapy with low-dose interleukin-2: rationale for prevention of immune-deficiency-associated cancer.

PURPOSE: Congenital, acquired, and some iatrogenically induced immune deficiencies are characterized by an increased incidence of viral-associated cancers. Preclinical and clinical studies were conducted to understand the pathogenesis of immune-deficiency-associated cancer and its response to low-dose recombinant interleukin-2 (rIL-2) therapy, with the ultimate goal of applying this or other immune therapy in the treatment or prevention of immune-deficiency-associated lymphoma. METHODS: We have used the severe combined immune-deficient (SCID) mouse engrafted with human peripheral blood lymphocytes (PBL) from healthy Epstein-Barr virus seropositive donors to study the pathogenesis of malignant B-cell lymphoproliferative disease that commonly occurs in some immune-deficient patients. In this chimeric human (hu)-PBL-SCID mouse model, administration of daily low-dose rIL-2 interacts with murine natural killer cells and human CD8+ T cells to prevent the outgrowth of human Epstein-Barr virus lymphoproliferative disease. We have utilized the information gained from this chimeric mouse model to perform a phase I study of daily, subcutaneous, low-dose rIL-2 therapy in patients with both acquired immune deficiency syndrome (AIDS) and cancer. RESULTS: Plasma concentrations of rIL-2 were achieved in vivo comparable to those seen in our hu-PBL-SCID model, in the absence of significant (grade 3) clinical toxicity or an increase in the plasma human immune deficiency virus (HIV) RNA level. Significant expansion in human cells, particularly the CD3-CD56bright natural killer cell subset, resulted after 6 weeks of therapy. Results of the hu-PBL-SCID mouse model and the phase I study have led to a national trial of low-dose rIL-2 therapy in AIDS-associated lymphoma. CONCLUSION: Daily low-dose rIL-2 therapy may be effective in treating or preventing AIDS-associated lymphoma without amplifying HIV replication.

Does previous surgery influence the asymmetric distribution of endometriotic lesions?

Our objective was to investigate the role of previous abdominal-pelvic surgery in the asymmetric distribution of pelvic endometriosic lesions. This was a retrospective study carried out at the Clinic of Obstetrics and Gynecology, University of Ancona, Italy, and included 238 patients with histological confirmation of endometriosis. The interventions were surgical treatment, at laparoscopy or laparotomy, for pelvic pain and endometriosis. The main outcome measure(s) were endometriotic lesions and adhesions in the pelvis found during surgery and the dinical records of the patients. We found unilateral lesions in 149 patients (62.6%): the right side of the pelvis affected in 55 patients (36.9%) and the left side in 94 patients (63.1%) (p < 0.01). In the group of patients who had undergone previous abdominal surgery, we found lesions on the right side in 26 cases (32.5%), and on the left in 54 cases (67.5%) (p < 0.01). We found that the patients who had undergone previous abdominal surgery had significantly more adhesions than those with no previous surgery (80/116 vs. 73/122, p = 0. 002). As a new finding, we have demonstrated that the left side of asymmetric distribution of intrapelvic macroscopic lesions is preserved and more evident in patients with previous abdominal surgery, including previous appendectomy. These data seem to be in agreement with our previous supposition of a possible interaction between previous abdominal surgery and the mechanisms of endometriosis development.

[Presenting forms and diagnostic procedures in gigantic hemangioma of the liver]. / Formas de presentación y procedimientos diagnósticos del hemangioma hepático gigante.

We present eleven patients diagnosed of giant hepatic hemangioma in the last 20 years. The diagnosis was confirmed in all the cases during laparoscopy or laparotomy. The mean age of the patients was 44.9 +/- 8.99; nine of them were women. Only two of the patients complained of abdominal pain. Five patients showed abnormal liver function tests; the most common finding was increased levels of alkaline phosphatase. We have reviewed the diagnostic tools employed: isotopic study of the liver with 99Tc, and labeled erythrocytes, abdominal ultrasonography, CAT, hepatic arteriography, laparoscopy, laparotomy and liver biopsy. Usually we employed more than one of these diagnostic methods. In the last years there has been a shift to employ less invasive procedures.

Expression of the zeta protein subunit in CD3- NK effectors derived from human thymus.

The origin, lineage derivation, and sites of human natural killer (NK) cell differentiation are presently unresolved. The vast majority of NK cells found in peripheral blood have surface membrane expression of CD2 and CD16. Both antigens trigger activation pathways which require the zeta protein, a signal-transducing subunit of the CD3-T cell receptor (CD3-TCR) complex which is found as an isolated homodimer (zeta-zeta) or heterodimer (zeta-Fc epsilon RI gamma) in human NK cells. Unlike NK cells found in adult peripheral blood, NK cells derived in vitro from human CD34+ hematopoietic progenitor cells lack CD2 and CD16, and those found in fetal liver constitutively express CD3 epsilon and delta proteins. However, NK effectors derived in vitro from immature human CD3- thymocytes show striking phenotypic and functional similarities to adult human NK cells. In this report, we characterize zeta protein expression in CD3- thymocytes following short-term culture in recombinant (r)IL-2. CD3-CD56+ thymocyte NK effectors express the zeta protein as a disulphide-linked homodimer of 32 kDa, yet lack other protein components of the CD3-TCR complex. Both CD16+ and CD16- populations were found to express zeta, and within the CD16+ fraction, zeta is physically associated with CD16. These data provide evidence of additional similarities between adult peripheral blood NK cells and CD3-CD56+ NK effectors derived from human thymocytes, and suggest that under these experimental conditions, human NK cells can arise from early thymic precursors.