Fermentation Gone Wild: A Biochemistry Laboratory Experiment.

An experiment for the upper-level biochemistry laboratory is described in which students isolate a wild yeast from environmental sources and characterize the strain for its potential in the brewing industry. In addition to providing valuable experience in important biochemical techniques, this study also illustrates key principles of bioprospecting, the search for new biological sources with potential commercial or scientific value. By foraging for yeast in the wild, students explore the microbial diversity of their local environment and potentially find untapped sources of yeast that produce novel flavors and aromas. Overall, students engage with hands-on experience in bioprospecting, allowing them to appreciate the value of exploring biological diversity and its potential applications in the brewing industry.

Species Widely Distributed in Halophilic Archaea Exhibit Opsin-Mediated Inhibition of Bacterioruberin Biosynthesis.

Halophilic Archaea are a distinctive pink color due to a carotenoid pigment called bacterioruberin. To sense or utilize light, many halophilic Archaea also produce rhodopsins, complexes of opsin proteins with a retinal prosthetic group. Both bacterioruberin and retinal are synthesized from isoprenoid precursors, with lycopene as the last shared intermediate. We previously described a regulatory mechanism by which Halobacterium salinarum bacterioopsin and Haloarcula vallismortis cruxopsin inhibit bacterioruberin synthesis catalyzed by lycopene elongase. In this work, we found that opsins in all three major Halobacteria clades inhibit bacterioruberin synthesis, suggesting that this regulatory mechanism existed in the common Halobacteria ancestor. Halophilic Archaea, which are generally heterotrophic and aerobic, likely evolved from an autotrophic, anaerobic methanogenic ancestor by acquiring many genes from Bacteria via lateral gene transfer. These bacterial "imports" include genes encoding opsins and lycopene elongases. To determine if opsins from Bacteria inhibit bacterioruberin synthesis, we tested bacterial opsins and found that an opsin from Curtobacterium, in the Actinobacteria phylum, inhibits bacterioruberin synthesis catalyzed by its own lycopene elongase, as well as that catalyzed by several archaeal enzymes. We also determined that the lycopene elongase from Halococcus salifodinae, a species from a family of Halobacteria lacking opsin homologs, retained the capacity to be inhibited by opsins. Together, our results indicate that opsin-mediated inhibition of bacterioruberin biosynthesis is a widely distributed mechanism found in both Archaea and Bacteria, possibly predating the divergence of the two domains. Further analysis may provide insight into the acquisition and evolution of the genes and their host species.IMPORTANCE All organisms use a variety of mechanisms to allocate limited resources to match their needs in their current environment. Here, we explore how halophilic microbes use a novel mechanism to allow efficient production of rhodopsin, a complex of an opsin protein and a retinal prosthetic group. We previously demonstrated that Halobacterium salinarum bacterioopsin directs available resources toward retinal by inhibiting synthesis of bacterioruberin, a molecule that shares precursors with retinal. In this work, we show that this mechanism can be carried out by proteins from halophilic Archaea that are not closely related to H. salinarum and those in at least one species of Bacteria Therefore, opsin-mediated inhibition of bacterioruberin synthesis may be a highly conserved, ancient regulatory mechanism.

Opsin-Mediated Inhibition of Bacterioruberin Synthesis in Halophilic Archaea.

Halophilic archaea often inhabit environments with limited oxygen, and many produce ion-pumping rhodopsin complexes that allow them to maintain electrochemical gradients when aerobic respiration is inhibited. Rhodopsins require a protein, an opsin, and an organic cofactor, retinal. We previously demonstrated that in Halobacterium salinarum, bacterioopsin (BO), when not bound by retinal, inhibits the production of bacterioruberin, a biochemical pathway that shares intermediates with retinal biosynthesis. In this work, we used heterologous expression in a related halophilic archaeon, Haloferax volcanii, to demonstrate that BO is sufficient to inhibit bacterioruberin synthesis catalyzed by the H. salinarum lycopene elongase (Lye) enzyme. This inhibition was observed both in liquid culture and in a novel colorimetric assay to quantify bacterioruberin abundance based on the colony color. Addition of retinal to convert BO to the bacteriorhodopsin complex resulted in a partial rescue of bacterioruberin production. To explore if this regulatory mechanism occurs in other organisms, we expressed a Lye homolog and an opsin from Haloarcula vallismortis in H. volcaniiH. vallismortis cruxopsin-3 expression inhibited bacterioruberin synthesis catalyzed by H. vallismortis Lye but had no effect when bacterioruberin synthesis was catalyzed by H. salinarum or H. volcanii Lye. Conversely, H. salinarum BO did not inhibit H. vallismortis Lye activity. Together, our data suggest that opsin-mediated inhibition of Lye is potentially widespread and represents an elegant regulatory mechanism that allows organisms to efficiently utilize ion-pumping rhodopsins obtained through lateral gene transfer.IMPORTANCE Many enzymes are complexes of proteins and nonprotein organic molecules called cofactors. To ensure efficient formation of functional complexes, organisms must regulate the production of proteins and cofactors. To study this regulation, we used bacteriorhodopsin from the archaeon Halobacterium salinarum Bacteriorhodopsin consists of the bacterioopsin protein and a retinal cofactor. In this article, we further characterize a novel regulatory mechanism in which bacterioopsin promotes retinal production by inhibiting a reaction that consumes lycopene, a retinal precursor. By expressing H. salinarum genes in a different organism, Haloferax volcanii, we demonstrated that bacterioopsin alone is sufficient for this inhibition. We also found that an opsin from Haloarcula vallismortis has inhibitory activity, suggesting that this regulatory mechanism might be found in other organisms.

Intracellular trafficking and glycobiology of TbPDI2, a stage-specific protein disulfide isomerase in Trypanosoma brucei.

Trypanosoma brucei protein disulfide isomerase 2 (TbPDI2) is a bloodstream stage-specific lumenal endoplasmic reticulum (ER) glycoprotein. ER localization is dependent on the TbPDI2 C-terminal tetrapeptide (KQDL) and is mediated by TbERD2, an orthologue of the yeast ER retrieval receptor. Consistent with this function, TbERD2 localizes prominently to ER exit sites, and RNA interference (RNAi) knockdown results in specific secretion of a surrogate ER retention reporter, BiPN:KQDL. TbPDI2 is highly N-glycosylated and is reactive with tomato lectin, suggesting the presence of poly-N-acetyllactosamine modifications, which are common on lyso/endosomal proteins in trypanosomes but are inconsistent with ER localization. However, TbPDI2 is reactive with tomato lectin immediately following biosynthesis-far too rapidly for transport to the Golgi compartment, the site of poly-N-acetyllactosamine addition. TbPDI2 also fails to react with Erythrina cristagalli lectin, confirming the absence of terminal N-acetyllactosamine units. We propose that tomato lectin binds the Manß1-4GlcNAcß1-4GlcNAc trisaccharide core of paucimannose glycans on both newly synthesized and mature TbPDI2. Consistent with this proposal, α-mannosidase treatment renders oligomannose N-glycans on the T. brucei cathepsin L orthologue TbCatL reactive with tomato lectin. These findings resolve contradictory evidence on the location and glycobiology of TbPDI2 and provide a cautionary note on the use of tomato lectin as a poly-N-acetyllactosamine-specific reagent.

Q-TWiST analysis of ixabepilone in combination with capecitabine on quality of life in patients with metastatic breast cancer.

BACKGROUND: Combination therapy with ixabepilone and capecitabine (cape) is approved for use in patients with locally advanced/metastatic breast cancer that is resistant to treatment with anthracyclines or taxanes. The current study evaluated the trade-off between quality and quantity of life using quality-adjusted time without symptoms or toxicity (Q-TWiST) outcomes. METHODS: Within the trial, 752 women were randomly assigned to receive either the combination of ixabepilone and cape (once every 21 days) or cape alone (on days 1-14). The area under the survival curve was partitioned into 3 health states: toxicity (TOX), time without symptoms of disease progression or toxicity, and recurrence (relapse [REL]). The mean time in each health state was weighted by a range of utilities and summed to estimate quality-adjusted survival (QAS). Patient-reported outcomes were also evaluated using the Functional Assessment of Cancer Therapy (FACT)-Breast Symptom Index (FBSI). RESULTS: A statistically significant difference between groups with regard to change from baseline FBSI scores favoring the cape group was observed (P = .0002), but no differences were observed after adjusting for deaths in the analysis. All combinations of utilities for REL and TOX resulted in an observed difference in QAS favoring combination therapy. Differences were found to be statistically significant for comparisons, with higher tolerance for TOX. QAS was found to be greater for the combination therapy group (42.2 weeks vs 38.4 weeks), assuming the base case scenario of utility equal to 0.5 for both TOX and REL (P = .0227). CONCLUSIONS: The Q-TWiST analysis supports a positive benefit-risk ratio for the combination of ixabepilone plus cape in patients with advanced/metastatic breast cancer that is refractory to anthracyclines and taxanes versus cape alone, despite the potential for added toxicities with combination therapy.

Ixabepilone-associated peripheral neuropathy: data from across the phase II and III clinical trials.

PURPOSE: Dose-limiting neuropathy is a major adverse event associated with most of the microtubule-stabilizing agent-based chemotherapy regimens. Ixabepilone, a semisynthetic analogue of the natural epothilone B, has activity against a wide range of tumor types. Peripheral neuropathy (PN), associated with ixabepilone treatment, is usually mild to moderate, predominantly sensory and cumulative. Preclinical studies demonstrate that ixabepilone and taxanes produce a similar neurotoxicity profile. METHODS: We searched databases of phase II/III clinical trials involving patients receiving ixabepilone as a monotherapy or in combination with capecitabine for incidences of neuropathy. Potential risk factors for grade 3/4 PN were identified by a Cox regression analysis on a dataset of 1,540 patients with different tumor types across multiple studies. RESULTS: Rates for incidence of ixabepilone-induced severe PN (Common Terminology Criteria for Adverse Events grade 3/4) ranged from 1% in early untreated breast cancer up to 24% in heavily pretreated metastatic breast cancer; grade 4 PN was rare (≤ 1%). Common symptoms included numbness, paresthesias, and sometimes dysesthesias. Cox regression analysis identified only preexisting neuropathy as a risk factor for increased ixabepilone-associated PN. The management of PN has been primarily through dose adjustments (dose delays and/or dose reduction). Patients had resolution of their neuropathy within a median time of 5 to 6 weeks. CONCLUSIONS: PN is a dose-limiting toxicity associated with ixabepilone treatment, is reversible in most patients, and can be managed with dose reduction and delays.

Bacterioopsin-mediated regulation of bacterioruberin biosynthesis in Halobacterium salinarum.

Integral membrane protein complexes consisting of proteins and small molecules that act as cofactors have important functions in all organisms. To form functional complexes, cofactor biosynthesis must be coordinated with the production of corresponding apoproteins. To examine this coordination, we study bacteriorhodopsin (BR), a light-induced proton pump in the halophilic archaeon Halobacterium salinarum. This complex consists of a retinal cofactor and bacterioopsin (BO), the BR apoprotein. To examine possible novel regulatory mechanisms linking BO and retinal biosynthesis, we deleted bop, the gene that encodes BO. bop deletion resulted in a dramatic increase of bacterioruberins, carotenoid molecules that share biosynthetic precursors with retinal. Additional studies revealed that bacterioruberins accumulate in the absence of BO regardless of the presence of retinal or BR, suggesting that BO inhibits bacterioruberin biosynthesis to increase the availability of carotenoid precursors for retinal biosynthesis. To further examine this potential regulatory mechanism, we characterized an enzyme, encoded by the lye gene, that catalyzes bacterioruberin biosynthesis. BO-mediated inhibition of bacterioruberin synthesis appears to be specific to the H. salinarum lye-encoded enzyme, as expression of a lye homolog from Haloferax volcanii, a related archaeon that synthesizes bacterioruberins but lacks opsins, resulted in bacterioruberin synthesis that was not reduced in the presence of BO. Our results provide evidence for a novel regulatory mechanism in which biosynthesis of a cofactor is promoted by apoprotein-mediated inhibition of an alternate biochemical pathway. Specifically, BO accumulation promotes retinal production by inhibiting bacterioruberin biosynthesis.

Ixabepilone plus capecitabine in metastatic breast cancer patients with reduced performance status previously treated with anthracyclines and taxanes: a pooled analysis by performance status of efficacy and safety data from 2 phase III studies.

Patients with metastatic breast cancer (MBC) previously treated with anthracyclines and taxanes often have decreased performance status secondary to extensive tumor involvement. Here, we report the pooled analysis of efficacy and safety data from two similarly designed phase III studies to provide a more precise estimate of benefit of ixabepilone plus capecitabine in MBC patients with Karnofsky's performance status (KPS) 70-80. Across the studies, anthracycline/taxane-pretreated MBC patients were randomized to receive ixabepilone plus capecitabine or capecitabine alone. Individual patient data for KPS 70-80 subset (n = 606) or KPS 90-100 subset (n = 1349) from the two studies were pooled by treatment. Analysis included overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and safety. In patients with reduced performance status (KPS 70-80), ixabepilone plus capecitabine was associated with improvements in OS (median: 12.3 vs. 9.5 months; HR, 0.75; P = 0.0015), PFS (median: 4.6 vs. 3.1 months; HR, 0.76; P = 0.0021) and ORR (35 vs. 19%) over capecitabine alone. Corresponding results in patients with high performance status (KPS 90-100) were median OS of 16.7 versus 16.2 months (HR, 0.98; P = 0.8111), median PFS of 6.0 versus 4.4 months (HR, 0.58; P = 0.0009), and ORR of 45 versus 28%. The safety profile of combination therapy was similar between the subgroups. Ixabepilone plus capecitabine appeared to show superior efficacy compared to capecitabine alone in MBC patients previously treated with anthracyclines and taxanes, regardless of performance status, with a possible OS benefit favoring KPS 70-80 patients (ClinicalTrials.gov identifiers: NCT00080301 and NCT00082433).

Analysis of overall survival from a phase III study of ixabepilone plus capecitabine versus capecitabine in patients with MBC resistant to anthracyclines and taxanes.

Limited proven treatment options exist for patients with metastatic breast cancer (MBC) resistant to anthracycline and taxane treatment. Ixabepilone, a novel semisynthetic analog of epothilone B, has demonstrated single-agent activity in MBC resistant to anthracyclines and taxanes. In combination with capecitabine in a phase III trial (CA163-046) in this setting, ixabepilone prolonged progression-free survival and increased objective response rate relative to capecitabine (Thomas et al. J Clin Oncol 25:5210-5217, 2007). Here, we report the results of overall survival (OS), a secondary efficacy endpoint from the CA163-046 trial. Seven hundred fifty-two patients with MBC resistant to anthracyclines and taxanes were randomized to ixabepilone (40 mg/m(2) intravenously on day 1 of a 21-day cycle) plus capecitabine (2,000 mg/m(2) orally on days 1 through 14 of a 21-day cycle) or capecitabine alone (2,500 mg/m(2) on the same schedule). Patients receiving ixabepilone plus capecitabine treatment had a median survival of 12.9 months compared to 11.1 months for patients receiving capecitabine alone (HR = 0.9; 95%CI: 077-1.05; P = 0.19). This observed increase in median OS favored the combination; however, the difference was not statistically significant. Predefined subset analyses showed a clinically meaningful increase in OS in KPS 70-80 patients receiving ixabepilone plus capecitabine (HR = 0.75; 95% CI: 0.58-0.98). Ixabepilone plus capecitabine did not show a significant improvement in survival compared to capecitabine alone in patients with MBC resistant to anthracyclines and taxanes. The observed differences in survival favored the combination arm. A clinical benefit was also seen in patients in the KPS 70-80 subgroup.

The LAMP-like protein p67 plays an essential role in the lysosome of African trypanosomes.

RNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific approximately 90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (approximately 20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four- to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH--chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.

Safety of repeated administrations of ixabepilone given as a 3-hour infusion every other week in combination with irinotecan in patients with advanced malignancies.

Epothilones are active tubulin-interacting agents that warrant combinations in clinical studies. This phase I combination study explored ixabepilone administered as a 3-h infusion followed by a 90-minute infusion irinotecan, on days 1 and 14 of every 28-day cycle. Forty-one patients received doses of ixabepilone and irinotecan ranging from 15-30 mg/m(2) and 120-180 mg/m(2) every 2 weeks for a total of 173 cycles, respectively. Dose limiting toxicities reported at doses 25 mg/m(2) ixabepilone and 180 mg/m(2) irinotecan consisted of acute grade 3 diarrhoea and asthenia, eventually associated with neutropenia and sepsis, and/or delayed grade 3 peripheral neuropathy. Therefore, the recommended doses were 20 mg/m(2) ixabepilone and 180 mg/m(2) irinotecan. At this dose level, acute side effects were neutropenia, anaemia, nausea-vomiting, diarrhoea, asthenia, and alopecia. Delayed neuropathy was mostly restricted to reversible grade I-II. Pharmacokinetic data suggested no drug-drug interaction. Five objective responses were observed in four patients with lung cancer and one unknown primary epidermoid carcinoma patient. In conclusion, toxicity including peripheral neuropathy was manageable at the recommended doses of 20 mg/m(2) ixabepilone combined with 180 mg/m(2) irinotecan on days 1 and 14 every 28 days. Promising antitumour activity was observed in patients with platinum-pretreated lung cancer.

Phase I/II study of ixabepilone plus capecitabine in anthracycline-pretreated/resistant and taxane-resistant metastatic breast cancer.

PURPOSE: The aim of this study was to determine the safety, maximum tolerated dose (MTD), recommended phase II dose, and efficacy of the epothilone B analogue ixabepilone plus capecitabine in anthracycline-pretreated/ resistant and taxane-resistant metastatic breast cancer (MBC). PATIENTS AND METHODS: A total of 106 patients were enrolled. The study consisted of a dose-escalation phase (phase I) and a tumor response rate evaluation phase (phase II). Seventy-four patients were treated in phase I with schedule A (ixabepilone 40 mg/m2 intravenously on day 1 plus capecitabine 1650-2000 mg/m2 on days 1-14 of a 21-day cycle) or schedule B (ixabepilone 8-10 mg/m2 on days 1-3 plus capecitabine 1650 mg/m2 on days 1-14 of a 21- day cycle). RESULTS: No dose-limiting toxicities (DLTs) were observed in the 8/1650 mg/m2 and 10/1650 mg/m2 cohorts; 1 of 30 patients in the 40/1650 mg/m2 cohort and 2 of 30 patients in the 40/2000 mg/m2 cohort had a DLT consisting of grade 3 plantar-palmar erythrodysesthesia (PPE). The 40/2000 mg/m2 dose was defined as the MTD for schedule A, and a total of 62 patients were treated for the phase II portion of the trial, which examined tumor response. The objective response rate was 30%, median time-to-response was 6 weeks, median duration of response was 6.9 months, and median progression-free survival was 3.8 months. Grade 3/4 treatment-related events in phase II included fatigue (34%), PPE (34%), myalgia (23%), nausea (16%), peripheral neuropathy (19%), and diarrhea/vomiting (10%). Grades 3/4 neutropenia (69%) and leukopenia (55%) were managed primarily by dose reduction/treatment interruption. CONCLUSION: Ixabepilone plus capecitabine demonstrated clinical activity and an acceptable safety profile in patients with anthracycline-pretreated/resistant and taxane-resistant MBC. Ixabepilone was recently approved in the United States for the treatment of resistant/refractory locally advanced or MBC.

Effect of a Patient and Clinician Communication-Priming Intervention on Patient-Reported Goals-of-Care Discussions Between Patients With Serious Illness and Clinicians: A Randomized Clinical Trial.

Importance: Clinician communication about goals of care is associated with improved patient outcomes and reduced intensity of end-of-life care, but it is unclear whether interventions can improve this communication. Objective: To evaluate the efficacy of a patient-specific preconversation communication-priming intervention (Jumpstart-Tips) targeting both patients and clinicians and designed to increase goals-of-care conversations compared with usual care. Design, Setting, and Participants: Multicenter cluster-randomized trial in outpatient clinics with physicians or nurse practitioners and patients with serious illness. The study was conducted between 2012 and 2016. Interventions: Clinicians were randomized to the bilateral, preconversation, communication-priming intervention (n = 65) or usual care (n = 67), with 249 patients assigned to the intervention and 288 to usual care. Main Outcomes and Measures: The primary outcome was patient-reported occurrence of a goals-of-care conversation during a target outpatient visit. Secondary outcomes included clinician documentation of a goals-of-care conversation in the medical record and patient-reported quality of communication (Quality of Communication questionnaire [QOC]; 4-indicator latent construct) at 2 weeks, as well as patient assessments of goal-concordant care at 3 months and patient-reported symptoms of depression (8-item Patient Health Questionnaire; PHQ-8) and anxiety (7-item Generalized Anxiety Disorder survey; GAD-7) at 3 and 6 months. Analyses were clustered by clinician and adjusted for confounders. Results: We enrolled 132 of 485 potentially eligible clinicians (27% participation; 71 women [53.8%]; mean [SD] age, 47.1 [9.6] years) and 537 of 917 eligible patients (59% participation; 256 women [47.7%]; mean [SD] age, 73.4 [12.7] years). The intervention was associated with a significant increase in a goals-of-care discussion at the target visit (74% vs 31%; P < .001) and increased medical record documentation (62% vs 17%; P < .001), as well as increased patient-rated quality of communication (4.6 vs 2.1; P = .01). Patient-assessed goal-concordant care did not increase significantly overall (70% vs 57%; P = .08) but did increase for patients with stable goals between 3-month follow-up and last prior assessment (73% vs 57%; P = .03). Symptoms of depression or anxiety were not different between groups at 3 or 6 months. Conclusions and Relevance: This intervention increased the occurrence, documentation, and quality of goals-of-care communication during routine outpatient visits and increased goal-concordant care at 3 months among patients with stable goals, with no change in symptoms of anxiety or depression. Understanding the effect on subsequent health care delivery will require additional study. Trial Registration: ClinicalTrials.gov identifier: NCT01933789.

Targeting KIT on innate immune cells to enhance the antitumor activity of checkpoint inhibitors.

Innate immune cells such as mast cells and myeloid-derived suppressor cells are key components of the tumor microenvironment. Recent evidence indicates that levels of myeloid-derived suppressor cells in melanoma patients are associated with poor survival to checkpoint inhibitors. This suggests that targeting both the innate and adaptive suppressive components of the immune system will maximize clinical benefit and elicit more durable responses in cancer patients. Preclinical data suggest that targeting signaling by the receptor tyrosine kinase KIT, particularly on mast cells, may modulate innate immune cell numbers and activity in tumors. Here, we review data highlighting the importance of the KIT signaling in regulating antitumor immune responses and the potential benefit of combining selective KIT inhibitors with immune checkpoint inhibitors.

The development of immunomodulatory monoclonal antibodies as a new therapeutic modality for cancer: the Bristol-Myers Squibb experience.

The discovery and increased understanding of the complex interactions regulating the immune system have contributed to the pharmacologic activation of antitumor immunity. The activity of effector cells, such as T and NK cells, is regulated by an array of activating and attenuating receptors and ligands. Agents that target these molecules can modulate immune responses by exerting antagonistic or agonistic effects. Several T- or NK-cell modulators have entered clinical trials, and two have been approved for use. Ipilimumab (Yervoy®, Bristol-Myers Squibb) and nivolumab (OPDIVO, Ono Pharmaceutical Co., Ltd./Bristol-Myers Squibb) were approved for the treatment of metastatic melanoma, in March 2011 in the United States, and in July 2014 in Japan, respectively. The clinical activity of these two antibodies has not been limited to tumor types considered sensitive to immunotherapy, and promising activity has been reported in other solid and hematologic tumors. Clinical development of ipilimumab and nivolumab has presented unique challenges in terms of safety and efficacy, requiring the establishment of new evaluation criteria for adverse events and antitumor effects. Guidelines intended to help oncologists properly manage treatment in view of these non-traditional features have been implemented. The introduction of this new modality of cancer treatment, which is meant to integrate with or replace the current standards of care, requires additional efforts in terms of optimization of treatment administration, identification of biomarkers and application of new clinical trial designs. The availability of immune modulators with different mechanisms of action offers the opportunity to establish immunological combinations as new standards of care.

Ixabepilone plus capecitabine in advanced breast cancer patients with early relapse after adjuvant anthracyclines and taxanes: a pooled subset analysis of two phase III studies.

BACKGROUND: Metastatic breast cancer (MBC) patients with rapid disease relapse after neo/adjuvant chemotherapy including anthracyclines and taxanes have limited treatment options and their efficacy is marginal. Two phase III studies compared ixabepilone plus capecitabine vs. capecitabine alone as first-line treatment in MBC patients pretreated with anthracyclines and taxanes in the neo/adjuvant setting. Here we report the efficacy and safety of these treatments in a prespecified subset of patients whose disease relapsed within 12 months. PATIENTS AND METHODS: Of 1973 patients across two studies, 293 relapsed within 12 months of neo/adjuvant treatment and received ixabepilone plus capecitabine (n = 149) or capecitabine alone (n = 144) as first-line chemotherapy for MBC. Analysis included progression-free survival (PFS), overall survival (OS), objective response rate (ORR) and toxicity. RESULTS: In 293 patients, ixabepilone plus capecitabine, as compared to capecitabine alone, increased PFS (median: 5.6 months vs. 2.8 months; hazard ratio, 0.58; p < 0.0001), ORR (46% vs. 24%) and OS (median: 15.1 months vs. 12.5 months; hazard ratio, 0.84; p = 0.208). Major toxicities of this regimen included neuropathy, neutropenia and hand-foot syndrome, but were manageable. CONCLUSIONS: Patients with breast cancer with early relapse following neo/adjuvant treatment with anthracyclines and taxanes may benefit from ixabepilone plus capecitabine. (ClinicalTrials.gov identifiers: NCT00080301 and NCT00082433.).

Randomized phase III trial of ixabepilone plus capecitabine versus capecitabine in patients with metastatic breast cancer previously treated with an anthracycline and a taxane.

PURPOSE: We sought to determine whether the combination of ixabepilone plus capecitabine improved overall survival (OS) compared with capecitabine alone in patients with metastatic breast cancer (MBC) previously treated with anthracyclines and taxanes. PATIENTS AND METHODS: A total of 1,221 patients with MBC previously treated with anthracycline and taxanes were randomly assigned to ixabepilone (40 mg/m(2) intravenously on day 1) plus capecitabine (2,000 mg/m(2) orally on days 1 through 14) or capecitabine alone (2,500 mg/m(2) on the same schedule) given every 21 days. The trial was powered to detect a 20% reduction in the hazard ratio (HR) for death. RESULTS: There was no significant difference in OS between the combination and capecitabine monotherapy arm, the primary end point (median, 16.4 v 15.6 months; HR = 0.9; 95% CI, 078 to 1.03; P = .1162). The arms were well balanced with the exception of a higher prevalence of impaired performance status (Karnofsky performance status 70% to 80%) in the combination arm (32% v 25%). In a secondary Cox regression analysis adjusted for performance status and other prognostic factors, OS was improved for the combination (HR = 0.85; 95% CI, 0.75 to 0.98; P = .0231). In 79% of patients with measurable disease, the combination significantly improved progression-free survival (PFS; median, 6.2 v 4.2 months; HR = 0.79; P = .0005) and response rate (43% v 29%; P < .0001). Grade 3 to 4 neuropathy occurred in 24% treated with the combination, but was reversible. CONCLUSION: This study confirmed a previous trial demonstrating improved PFS and response for the ixabepilone-capecitabine combination compared with capecitabine alone, although this did not result in improved survival.

A combination of hand-held models and computer imaging programs helps students answer oral questions about molecular structure and function: a controlled investigation of student learning.

We conducted a controlled investigation to examine whether a combination of computer imagery and tactile tools helps introductory cell biology laboratory undergraduate students better learn about protein structure/function relationships as compared with computer imagery alone. In all five laboratory sections, students used the molecular imaging program, Protein Explorer (PE). In the three experimental sections, three-dimensional physical models were made available to the students, in addition to PE. Student learning was assessed via oral and written research summaries and videotaped interviews. Differences between the experimental and control group students were not found in our typical course assessments such as research papers, but rather were revealed during one-on-one interviews with students at the end of the semester. A subset of students in the experimental group produced superior answers to some higher-order interview questions as compared with students in the control group. During the interview, students in both groups preferred to use either the hand-held models alone or in combination with the PE imaging program. Students typically did not use any tools when answering knowledge (lower-level thinking) questions, but when challenged with higher-level thinking questions, students in both the control and experimental groups elected to use the models.

Phase I dose escalation study of weekly ixabepilone, an epothilone analog, in patients with advanced solid tumors who have failed standard therapy.

PURPOSE: To establish the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), safety and recommended Phase II dose of ixabepilone, administered weekly as an intravenous (IV) infusion to patients with solid tumors who have failed standard therapy. METHOD: This was an open-label, single-arm, Phase I, dose-escalation study. RESULTS: The MTD of ixabepilone [30-min, weekly IV infusion on a 21-day schedule (N = 33)] was established at 25 mg/m(2). Grade 3 fatigue was the DLT in 2/4 patients treated at 30 mg/m(2). Ixabepilone was well tolerated at the MTD. Myelosuppression was rare, with no Grade 3/4 neutropenia. Due to the potential for cumulative neurotoxicity, the protocol was amended to a 1-h infusion, weekly for 3 weeks with a 1-week break. No DLT occurred at starting doses of 15, 20 and 25 mg/m(2) on this modified schedule (N = 51), although overall toxicity was less at 15 and 20 mg/m(2) than 25 mg/m(2). Five patients (2 on the 30-min/21-day schedule and 3 on the 60-min/28-day schedule) achieved durable objective partial responses across a variety of tumor types. CONCLUSIONS: Ixabepilone had an acceptable safety profile at the MTD of 25 mg/m(2) (as a 30-min weekly infusion on a continuous 21-day schedule) and at 20 mg/m(2) (as a 1-h weekly infusion on a modified 28-day schedule). The clinical activity and acceptable tolerability profile warrant further single- or combination-agent evaluation.

Phase I study of the novel epothilone analog ixabepilone (BMS-247550) in patients with advanced solid tumors and lymphomas.

PURPOSE: To establish the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), safety, pharmacokinetics, and pharmacodynamics of ixabepilone when administered as a 1-hour infusion every 3 weeks to patients with advanced solid tumors or relapsed/refractory non-Hodgkin's lymphoma. Dosing schedules of 40 mg/m2 and 50 mg/m2 over 3 hours were also evaluated. PATIENTS AND METHODS: Sixty-one patients were enrolled using an initial accelerated dose-escalation phase followed by a standard dose-escalation phase, with doses of ixabepilone ranging from 7.4 to 65 mg/m2. The pharmacokinetics of ixabepilone and two of its chemical degradation products were evaluated. Plasma pharmacodynamics were evaluated for both 1- and 3-hour infusions using an assay that measures the amount of endogenous tubulin in peripheral-blood mononuclear cells that exists in the polymerized versus the unpolymerized state. Response evaluation was performed every 6 weeks. RESULTS: The most common DLTs were neutropenia, stomatitis/pharyngitis, myalgia, and arthralgia. The MTD of ixabepilone as a 1-hour infusion every 3 weeks was established as 50 mg/m2. The maximum plasma concentration and area under the plasma concentration time curve appeared to increase less than proportionally to dose. Durable objective responses were seen in eight patients, including two complete responses. Five of the responders had experienced treatment failure with a taxane. CONCLUSION: The recommended dose of ixabepilone for the initiation of phase II studies on the basis of these results is 50 mg/m2 over 1 hour every 3 weeks. The promising efficacy and tolerability results demonstrated by ixabepilone in this study warrant its continued development.