CtIP is essential for early B cell proliferation and development in mice.

B cell development requires efficient proliferation and successful assembly and modifications of the immunoglobulin gene products. CtIP is an essential gene implicated in end resection and DNA repair. Here, we show that CtIP is essential for early B cell development but dispensable in naive B cells. CtIP loss is well tolerated in G1-arrested B cells and during V(D)J recombination, but in proliferating B cells, CtIP loss leads to a progressive cell death characterized by ATM hyperactivation, G2/M arrest, genomic instability, and 53BP1 nuclear body formation, indicating that the essential role of CtIP during proliferation underscores its stage-specific requirement in B cells. B cell proliferation requires phosphorylation of CtIP at T847 presumably by CDK, but not its interaction with CtBP or Rb or its nuclease activity. CtIP phosphorylation by ATM/ATR at T859 (T855 in mice) promotes end resection in G1-arrested cells but is dispensable for B cell development and class switch recombination, suggesting distinct roles for T859 and T847 phosphorylation in B cell development.

Optimal sequencing of ibrutinib, idelalisib, and venetoclax in chronic lymphocytic leukemia: results from a multicenter study of 683 patients.

Background: Ibrutinib, idelalisib, and venetoclax are approved for treating CLL patients in the United States. However, there is no guidance as to their optimal sequence. Patients and methods: We conducted a multicenter, retrospective analysis of CLL patients treated with kinase inhibitors (KIs) or venetoclax. We examined demographics, discontinuation reasons, overall response rates (ORR), survival, and post-KI salvage strategies. Primary endpoint was progression-free survival (PFS). Results: A total of 683 patients were identified. Baseline characteristics were similar in the ibrutinib and idelalisib groups. ORR to ibrutinib and idelalisib as first KI was 69% and 81%, respectively. With a median follow-up of 17 months (range 1-60), median PFS and OS for the entire cohort were 35 months and not reached. Patients treated with ibrutinib (versus idelalisib) as first KI had a significantly better PFS in all settings; front-line [hazard ratios (HR) 2.8, CI 1.3-6.3, P = 0.01], relapsed-refractory (HR 2.8, CI 1.9-4.1, P < 0.001), del17p (HR 2.0, CI 1.2-3.4, P = 0.008), and complex karyotype (HR 2.5, CI 1.2-5.2, P = 0.02). At the time of initial KI failure, use of an alternate KI or venetoclax had a superior PFS when compared with chemoimmunotherapy. Furthermore, patients who discontinued ibrutinib due to progression or toxicity had marginally improved outcomes if they received venetoclax (ORR 79%) versus idelalisib (ORR 46%) (PFS HR .6, CI.3-1.0, P = 0.06). Conclusions: In the largest real-world experience of novel agents in CLL, ibrutinib appears superior to idelalisib as first KI. Furthermore, in the setting of KI failure, alternate KI or venetoclax therapy appear superior to chemoimmunotherapy combinations. The use of venetoclax upon ibrutinib failure might be superior to idelalisib. These data support the need for trials testing sequencing strategies to optimize treatment algorithms.

Electroencephalogram signatures of loss and recovery of consciousness from propofol.

Unconsciousness is a fundamental component of general anesthesia (GA), but anesthesiologists have no reliable ways to be certain that a patient is unconscious. To develop EEG signatures that track loss and recovery of consciousness under GA, we recorded high-density EEGs in humans during gradual induction of and emergence from unconsciousness with propofol. The subjects executed an auditory task at 4-s intervals consisting of interleaved verbal and click stimuli to identify loss and recovery of consciousness. During induction, subjects lost responsiveness to the less salient clicks before losing responsiveness to the more salient verbal stimuli; during emergence they recovered responsiveness to the verbal stimuli before recovering responsiveness to the clicks. The median frequency and bandwidth of the frontal EEG power tracked the probability of response to the verbal stimuli during the transitions in consciousness. Loss of consciousness was marked simultaneously by an increase in low-frequency EEG power (<1 Hz), the loss of spatially coherent occipital alpha oscillations (8-12 Hz), and the appearance of spatially coherent frontal alpha oscillations. These dynamics reversed with recovery of consciousness. The low-frequency phase modulated alpha amplitude in two distinct patterns. During profound unconsciousness, alpha amplitudes were maximal at low-frequency peaks, whereas during the transition into and out of unconsciousness, alpha amplitudes were maximal at low-frequency nadirs. This latter phase-amplitude relationship predicted recovery of consciousness. Our results provide insights into the mechanisms of propofol-induced unconsciousness, establish EEG signatures of this brain state that track transitions in consciousness precisely, and suggest strategies for monitoring the brain activity of patients receiving GA.

Changing paradigms in the treatment of chronic lymphocytic leukemia.

Progress in our understanding of chronic lymphocytic leukemia and its treatment has resulted in a more tailored approach to patient management, with different therapeutic regimens for different patient populations. The current standard of care has evolved from single-agent therapy with chlorambucil or cyclophosphamide, through the introduction of purine analogs to the more recent introduction of chemoimmunotherapy. Selection of appropriate initial therapy should be based primarily on patient characteristics such as age, performance status and the expected clinical course of the leukemia based on established risk factors. Achieving a complete and durable response is the major goal for fit patients; chemoimmunotherapy with fludarabine, cyclophosphamide and rituximab would be advantageous. Alternatively, in unfit patients, controlling symptoms is the essential treatment goal and a regimen with a more favorable toxicity profile should be applied. This manuscript reviews the data that has lead to current treatment choices, advises on tailored therapies and discusses emerging trends. Data for this review was identified by a search of electronic information including Medline and PubMed databases, conference proceedings and trial registers. Critical analysis of extracted data was undertaken with attention to trial phase, treatment schedules and end points, including response rates, follow-up times, progression-free survival and overall survival.

Radioisotopic localization of (90)Yttrium-ibritumomab tiuxetan in patients with CD20+ non-Hodgkin's lymphoma.

PURPOSE: (90)Yttrium-ibritumomab-tiuxetan (Zevalin) is an effective treatment for relapsed or refractory low-grade, follicular, or transformed B-cell NHL. The purpose of this study is to assess whether tissue and cellular localization of (90)Y-ibritumomab-tiuxetan determined by autoradiography and radioactivity localized to tumor tissue might enhance our understanding of the mechanism of action of radioimmunotherapy. METHODS: Eight eligible patients had CD20+ NHL, a bulky peripheral lymph node, and were scheduled for (90)Y-ibritumomab-tiuxetan treatment. 2-Deoxy-2-[F-18]fluoro-D: -glucose-positron emission tomography/computed tomography (FDG-PET/CT) was performed prior to treatment and at 12 weeks after therapy for assessment of response. Bone marrow, lymph node, and blood samples were collected 114 +/- 3 h after 14.8 MBq/kg (90)Y-ibritumomab-tiuxetan and processed for histology, scintillation counting, and microscopic autoradiography. RESULTS: Pericellular membrane localization of (90)Y-ibritumomab-tiuxetan to lymphoma cells was observed by autoradiography in the involved areas of lymph node with absence of significant localization in histologically normal sections of bone marrow. Pericellular radioactivity and the highest quantitative radioactivity were observed in lymph node samples of responding patients. CONCLUSIONS: (90)Y-ibritumomab-tiuxetan localizes to the surface membrane of CD20+ lymphoma cells in affected lymph nodes. The patients with the highest quantitative concentration of radioactivity to the lymph node as determined by scintillation counting were observed to have a clinical and FDG-PET/CT response.

Cyclophosphamide followed by fludarabine for untreated chronic lymphocytic leukemia: a phase II SWOG TRIAL 9706.

B-cell chronic lymphocytic leukemia (CLL) accounts for 95% of chronic leukemia cases and 25% of all leukemia. Despite the prevalence of CLL, progress in its treatment has been only modest over the past three decades. Based upon the ability of fludarabine to produce high-grade remissions especially among patients with low initial tumor mass, and the ability of alkylators to reduce tumor mass, we hypothesized that sequential administration of a limited number of cycles of intermediate-dose cyclophosphamide followed by fludarabine could result in a larger percentage of patients with complete remissions (CRs). In all, 27 of the 49 eligible patients achieved overall responses of CR, unconfirmed complete remission (UCR), or PR, for a total response rate of 55% (95% confidence interval (CI) 40-69%). Considering the confounding medical issues of this patient population with advanced aggressive disease, the regimen was generally well tolerated. This study demonstrates that high-dose cyclophosphamide followed by fludarabine was relatively well tolerated in this group of advanced CLL patients. The study's criterion for testing whether the regimen is sufficiently effective to warrant further investigation was met: 14 (32%) of the first 44 eligible patients achieved CR or UCR.

Gene therapy for head and neck cancer using vaccinia virus expressing IL-2 in a murine model, with evidence of immune suppression.

We evaluated the efficiency of recombinant vaccinia virus expressing interleukin-2 (rvv-IL-2) as a tumor vaccine in an immunocompetent mouse model of head and neck squamous cell carcinoma (SCC VII/SF). Mice with five-day-old tumors in the floor of the mouth were treated with rvv-IL-2 by intratumoral injections. These treated mice survived longer (P <.03) than mice treated with control vaccines. Splenocytes, bone marrow, and lymph node cells from tumor-bearing mice responded poorly to concanavalin A stimulation, suggesting induction of immunosuppression. The rvv-IL-2 virus grew for 7 days in the tumor following intratumoral injection. We did not detect any virus particles in several normal organs following rvv-IL-2 injection. Comparison of expression levels of several potential immune inhibitory mediators between the tumors growing in mice and cultured tumor cells demonstrated higher expression of IL-10, GM-CSF, TGF-beta, and NO synthetase in tumors. These results suggested possible roles for these molecules in immunosuppression. We conclude that rvv-IL-2 has potential as a therapeutic vaccine for head and neck cancer and that it can be more effective provided the immunosuppression is reversed.

Murine monoclonal anti-idiotypic antibody as a surrogate antigen for human Her-2/neu.

The anti-idiotypic (Id) monoclonal antibody (MAb) 520C9-6b (IgG1k), raised in syngenic mice against the murine anti-Her2/neu MAb 520C9 (Ab1), functionally mimics a human Her2/neu epitope and serves as a surrogate for the protein antigen. Immunization of allogeneic C57BL/6 mice and rabbits with 520C9-6b (Ab2) induced anti-Her2/neu-specific antibodies that react with antigen-positive SKBr3 cells by ELISA and FACS analysis. The immune sera inhibited binding between Ab1 and Ab2 and vice versa (binding of Ab2 to Ab1), indicating that it was a true anti-anti-Id (Ab3) antibody. The Ab3 sera or purified Ab3 specifically lysed Her2/neu-positive SKBr3 cells, but no significant lysis was observed in antigen-negative LS174T cells in an antibody-dependent cellular cytotoxicity assay. An Id-specific cellular immune response was also demonstrated in an in vitro lymphocyte proliferation assay. Furthermore, a panel of tumor tissues and tumor cells was screened for the presence of the Her2/neu epitope by its reactivity with Ab1 and Ab3 using immunohistochemistry and FACS analysis. Identical results were obtained using either Ab1 or Ab3 (Ab1'). The data indicated that anti-Id 520C9-6b can induce Her2/neu-specific antibody in experimental animals and may serve as a potential network antigen for the treatment of patients bearing Her2/neu-positive tumors.

Immunotherapy for colorectal cancer.

Immunologic approaches to therapy for colorectal cancer have evolved substantially. In the past, patients were treated with nonspecific immune stimulants such as bacillus Calmette-Guérin (BCG). The current focus lies in targeting tumor-associated antigens. This is done either through passive immune therapy, with antibodies targeted directly to tumor cells, or by active immune therapy through vaccination with tumor cells, tumor cell lysates, peptides, carbohydrates, gene constructs encoding proteins, or anti-idiotype antibodies that mimic tumor-associated antigens. These different approaches to immunotherapy are reviewed.

The anti-idiotype vaccines for immunotherapy.

Certain anti-idiotypic antibodies that bind to the antigen-combining sites of antibodies can effectively mimic the three-dimensional structures and functions of the external antigens and can be used as surrogate antigens for active specific immunotherapy. Extensive studies in animal models have demonstrated the efficacy of these vaccines for triggering the immune system to induce specific and protective immunity against bacterial, viral and parasitic infections as well as tumors. Several monoclonal anti-idiotype antibodies that mimic distinct human tumor-associated antigens have been developed and characterized. Encouraging results have been obtained in recent clinical trials using these anti-idiotype antibodies as vaccines. In this article, we will review the current literature and discuss the potential of this novel therapeutic approach for various human cancers.

Adjuvant immunotherapy for melanoma and colorectal cancers.

Immune approaches to the therapy of colorectal cancer and melanoma have substantially evolved over the past years, from treating patients with nonspecific immune stimulants to a focus on the use of tumor-associated antigens (TAAs) either by passive immune therapy with antibodies targeted directly to tumor cells or by active immune therapy via vaccination with tumor cells, tumor cell lysates, peptides, carbohydrates, gene constructs encoding proteins, or anti-idiotype antibodies that mimic TAAs. We review the different immunotherapeutic approaches to the treatment of melanoma and colorectal cancers, and summarize relevant clinical trials.

Interim analysis of the use of the anti-idiotype breast cancer vaccine 11D10 (TriAb) in conjunction with autologous stem cell transplantation in patients with metastatic breast cancer.

The anti-idiotype monoclonal antibody breast cancer vaccine 11D10 (TriAb) was administered before and after autologous stem cell transplantation (ASCT) in 45 patients with metastatic breast cancer whose disease was responsive to conventional chemotherapy. Evidence of a positive anti-anti-idiotype antibody (Ab3) humoral response was noted at a median of 1.76 months post-ASCT (range, before ASCT-6 months) with this strategy. Maximal Ab3 levels and idiotype-specific T-cell proliferative responses were observed at a median of 3 and 4 months, respectively, after ASCT. The achievement of rapid immune responses after ASCT, during a known period of decreased immunoresponsiveness, opens the possibility of an additional antitumor effect at a time when the tumor burden is relatively small. Moreover, in this interim analysis, patients with the most vigorous humoral and cellular immune responses had a significant improvement in progression-free survival. Further follow-up and evaluation of this approach is warranted.

Construction and characterization of DNA vaccines encoding the single-chain variable fragment of the anti-idiotype antibody 1A7 mimicking the tumor-associated antigen disialoganglioside GD2.

Anti-idiotype antibody, 1A7, functionally mimics the tumor-associated antigen disialoganglioside GD2, which is overexpressed on the surface of a number of neuroectodermal tumors such as melanoma, neuroblastoma, soft tissue sarcoma, and small cell carcinoma of the lung. Immunization of mice with 1A7 generated the production of anti-GD2 antibodies. In a phase I clinical trial, immunization of patients with 1A7, mixed with the adjuvant QS21, demonstrated that 1A7 could act as a surrogate antigen for GD2 and induce strong humoral immune responses in advanced stage melanoma patients. DNA vaccines have recently been shown to invoke humoral as well as cellular responses in injected hosts against the transgene product. To evaluate the efficiency of DNA vaccines encoding anti-idiotype antibodies, we constructed expression plasmids encoding the variable heavy (VH) and variable light (VL) chains of 1A7. The plasmids were made in two configurations, expressing either the VH (pc1A7VHLnVL) or the VL (pc1A7VLLnVH) chain of 1A7 at the amino terminus, linked together by a 15-amino acid linker (Ln). In vitro transcription/translation assays and transfection of CHO-K1 cells with the plasmids demonstrated that a approximately 30-kDa protein was expressed by both configurations of the single-chain variable fragment. This protein can be specifically precipitated by monoclonal anti-GD2 antibody, 14G2a. Following intramuscular injection in mice, the plasmids were detectable in the injected tissues for at least 3 months and the injected plasmids actively transcribed the single-chain variable fragment 1A7 gene at the injected site. A single, intramuscular immunization of a group of C57BL/6 mice with pc1A7VLLnVH in phosphate-buffered saline induced humoral immune responses against 1A7 as well as GD2, the nominal antigen. Multiple immunizations, however, were required to elicit stronger immune responses.

Monoclonal antibody therapies for lymphomas.

Important advances in molecular biology and chelation chemistry have led to new and improved monoclonal antibody reagents. Rituximab (IDEC-C2B8) was approved by the United States Food and Drug Administration for relapsed CD20-positive lymphomas, and denileukin diftitox was approved (DAB389IL-2) for CTCL. Ibritumomab tiuxetan and iodine 131 anti-B1 have excellent activity and acceptable toxicity and likely higher responses than rituximab. These questions should be answered in phase III trials, and preliminary results from the rituximab versus ibritumomab tiuxetan trial suggest higher responses to the latter. Data on radiolabeled Lym-1, T101, LL2, and anti-Tac also look promising. The therapeutic role of immunotoxins has yet to be determined, but encouraging data with DAB389IL-2 and LMB-2 have been reported. Antibody conjugates with drugs, prodrugs, nonprotein toxins, and other agents are also under investigation. Bispecific antibodies for lymphoma may also have a future role in lymphoma therapy. It is anticipated that many of the major advances in lymphoma therapy will be antibody based.

Use of the anti-idiotype antibody vaccine TriAb after autologous stem cell transplantation in patients with metastatic breast cancer.

Between April 1997 and March 1998 we evaluated the immune response and outcome in 11 chemosensitive patients who were treated with the anti-idiotype antibody vaccine TriAb after recovery from intensive therapy and autologous stem cell transplant (ASCT). Triab was commenced after recovery from the acute effects of ASCT; a minimum interval of 1 month was required from completion of consolidation radiotherapy, if given. Nine patients (82%) manifest anti-anti-idiotype antibody (Ab3) responses post ASCT. The maximal Ab3 response was seen after a median of 10 doses (range 5-20), which corresponded to a median of 14 months (range 5-19) post ASCT. Evidence of a T cell proliferative response was seen in eight patients; the response was modest in most of these. At a median follow-up of 24 months (range 22-33) after ASCT, four patients are alive without evidence of disease progression. All four of these patients were in the subgroup with more vigorous immune responses. Subsequent efforts have been directed toward the achievement of higher levels of immune responses more rapidly post ASCT. Bone Marrow Transplantation (2000) 26, 729-735.

Anti-idiotype vaccine against cancer.

Immunization with anti-idiotype (Id) antibodies represents a novel new approach to active immunotherapy. Extensive studies in animal tumor models have demonstrated the efficacy of anti-Id vaccines in preventing tumor growth and curing mice with established tumor. We have developed and characterized several murine monoclonal anti-Id antibodies (Ab2) which mimic distinct human tumor-associated antigens (TAA) and can be used as surrogate antigens for triggering active anti-tumor immunity in cancer patients. Encouraging results have been obtained in recent clinical trials. In this article, we will review the existing literature and summarize our own findings showing the potential of this approach for various human cancers. We will also discuss where anti-Id vaccines may perform better than traditional antigen vaccines.

Counterpoint. Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine.

Anti-idiotype (Id) vaccine therapy has been tested and shown to be effective, in several animal models, for triggering the immune system to induce specific and protective immunity against bacterial, viral and parasitic infections. The administration of anti-Id antibodies as surrogate tumor-associated antigens (TAA) also represents another potential application of the concept of the Id network. Limited experience in human trials using anti-Id to stimulate immunity against tumors has shown promising results. In this "counter-point" article, we discuss our own findings showing the potential of anti-Id antibody vaccines to be novel therapeutic approaches to various human cancers and also discuss where anti-Id vaccines may perform better than traditional multiple-epitope antigen vaccines.

Clinical and immune responses in advanced melanoma patients immunized with an anti-idiotype antibody mimicking disialoganglioside GD2.

PURPOSE: To determine immune responses and toxicity to the anti-idiotype vaccine, as well as clinical responses and survival, we initiated a clinical trial for patients with advanced melanoma treated with an anti-idiotype antibody (TriGem) that mimics the disialoganglioside GD2. PATIENTS AND METHODS: Forty-seven patients with advanced melanoma received either 1-, 2-, 4-, or 8-mg doses of TriGem (Titan Pharmaceuticals Inc, South San Francisco, CA) mixed with QS-21 adjuvant (Aquila Biopharmaceuticals, Inc, Worcester, MA) 100 microg subcutaneously weekly for 4 weeks and then monthly until disease progression. Median age was 57 years, there were 32 men and 15 women, 43% of patients had undergone prior therapy for metastatic disease, 55% had disease confined to soft tissue, and 45% had visceral metastasis. RESULTS: Hyperimmune sera from 40 of 47 patients showed an anti-anti-idiotype (Ab3) response. Patient Ab3 was truly Ab1' because it specifically bound purified disialoganglioside GD2. The isotypic specificity of the Ab3 antibody consisted of predominantly immunoglobulin (Ig)G, and all IgG subclasses were represented. One patient had a complete response that persisted at 24 months, and 12 patients were stable from 14+ to 37+ months (median, 18+ months). Disease progression occurred in 32 patients on study from 1 to 17 months (median, 5.5 months), and 21 have died at 1 to 16 months (median, 6 months). The Kaplan-Meier-derived overall median survival has not been reached. Median survival has not been reached for the 26 patients with soft tissue disease only and was 13 months for 21 patients with visceral metastasis. Toxicity consisted of local reaction at the site of injection and mild fever and chills. CONCLUSION: TriGem has minimal toxicity and generates robust and specific IgG immune responses against GD2. Objective responses were minimal, but there may be a favorable impact on disease progression and survival that will require prospective randomized trials.