Efficacy and tolerability of trabectedin in elderly patients with sarcoma: subgroup analysis from a phase III, randomized controlled study of trabectedin or dacarbazine in patients with advanced liposarcoma or leiomyosarcoma.

Background: Treatment options for soft tissue sarcoma (STS) patients aged ≥65 years (elderly) can be limited by concerns regarding the increased risk of toxicity associated with standard systemic therapies. Trabectedin has demonstrated improved disease control in a phase III trial (ET743-SAR-3007) of patients with advanced liposarcoma or leiomyosarcoma after failure of anthracycline-based chemotherapy. Since previous retrospective analyses have suggested that trabectedin has similar safety and efficacy outcomes regardless of patient age, we carried out a subgroup analysis of the safety and efficacy observed in elderly patients enrolled in this trial. Patients and methods: Patients were randomized 2 : 1 to trabectedin (n = 384) or dacarbazine (n = 193) administered intravenously every-3-weeks. The primary end point was overall survival (OS); secondary end points were progression-free survival (PFS), time-to-progression, objective response rate (ORR), duration of response, symptom severity, and safety. A post hoc analysis was conducted in the elderly patient subgroup. Results: Among 131 (trabectedin = 94; dacarbazine = 37) elderly patients, disease characteristics were well-balanced and consistent with those of the total study population. Treatment exposure was longer in patients treated with trabectedin versus dacarbazine (median four versus two cycles, respectively), with a significantly higher proportion receiving prolonged therapy (≥6 cycles) in the trabectedin arm (43% versus 23%, respectively; P = 0.04). Elderly patients treated with trabectedin showed significantly improved PFS [4.9 versus 1.5 months, respectively; hazard ratio (HR)=0.40; P = 0.0002] but no statistically significant improvement in OS (15.1 versus 8.0 months, respectively; HR = 0.72; P = 0.18) or ORR (9% versus 3%, respectively; P = 0.43). The safety profile for elderly trabectedin-treated patients was comparable to that of the overall trabectedin-treated study population. Conclusions: This subgroup analysis of the elderly population of ET743-SAR-3007 suggests that elderly patients with STS and good performance status can expect clinical benefit from trabectedin similar to that observed in younger patients. Trial registration: www.clinicaltrials.gov, NCT01343277.

Doxorubicin plus the IGF-1R antibody cixutumumab in soft tissue sarcoma: a phase I study using the TITE-CRM model.

BACKGROUND: Insulin-like growth factor receptor (IGF-1R) has been studied as an oncologic target in soft tissue sarcoma (STS), but its role in sarcoma biology is unclear. Anti-IGF-1R antibody cixutumumab demonstrated acceptable toxicity but limited activity as a single agent in STS. We carried out a dose-escalation study of cixutumumab with doxorubicin to evaluate safety and dosing of the combination. PATIENTS AND METHODS: Eligible patients with advanced STS were treated with cixutumumab intravenously on days 1/8/15 at one of three dose levels (A: 1 mg/kg, B: 3 mg/kg, C: 6 mg/kg) with doxorubicin at 75 mg/m(2) as a 48 h infusion on day 1 of a 21 day cycle. After six cycles of the combination, patients could receive cixutumumab alone. The Time-to-Event Continual Reassessment Method was used to estimate the probability of dose-limiting toxicity (DLT) and to assign patients to the dose with an estimated probability of DLT≤20%. RESULTS: Between September 2008 and January 2012, 30 patients with advanced STS received a median of six cycles of therapy (range <1-22). Two DLTs were observed, grade 3 mucositis (dose level B) and grade 4 hyperglycemia (dose level C). Grade 2 and 3 reduced left ventricular ejection fraction was seen in three and two patients, respectively. Five partial responses were observed, and estimated progression-free survival was 5.3 months (95% confidence interval 3.0-6.3) in 26 response-assessable patients. Immunohistochemical staining of 11 available tumor samples for IGF-1R and phospho-IGF-1R was not significantly different among responders and non-responders, and serum analysis of select single-nucleotide polymorphisms did not predict for cardiotoxicity. CONCLUSION: The maximum tolerated dose was doxorubicin 75 mg/m(2) on day 1 and cixitumumab 6 mg/kg on days 1/8/15 of a 21 day cycle. Cardiac toxicity was observed and should be monitored in subsequent studies, which should be considered in STS only if a predictive biomarker of benefit to anti-IGF-1R therapy is identified. TRIAL REGISTRATION: ClinicalTrials.gov:NCT00720174.

Guidelines for time-to-event end point definitions in sarcomas and gastrointestinal stromal tumors (GIST) trials: results of the DATECAN initiative (Definition for the Assessment of Time-to-event Endpoints in CANcer trials)†.

BACKGROUND: The use of potential surrogate end points for overall survival, such as disease-free survival (DFS) or time-to-treatment failure (TTF) is increasingly common in randomized controlled trials (RCTs) in cancer. However, the definition of time-to-event (TTE) end points is rarely precise and lacks uniformity across trials. End point definition can impact trial results by affecting estimation of treatment effect and statistical power. The DATECAN initiative (Definition for the Assessment of Time-to-event End points in CANcer trials) aims to provide recommendations for definitions of TTE end points. We report guidelines for RCT in sarcomas and gastrointestinal stromal tumors (GIST). METHODS: We first carried out a literature review to identify TTE end points (primary or secondary) reported in publications of RCT. An international multidisciplinary panel of experts proposed recommendations for the definitions of these end points. Recommendations were developed through a validated consensus method formalizing the degree of agreement among experts. RESULTS: Recommended guidelines for the definition of TTE end points commonly used in RCT for sarcomas and GIST are provided for adjuvant and metastatic settings, including DFS, TTF, time to progression and others. CONCLUSION: Use of standardized definitions should facilitate comparison of trials' results, and improve the quality of trial design and reporting. These guidelines could be of particular interest to research scientists involved in the design, conduct, reporting or assessment of RCT such as investigators, statisticians, reviewers, editors or regulatory authorities.

Trabectedin is a feasible treatment for soft tissue sarcoma patients regardless of patient age: a retrospective pooled analysis of five phase II trials.

BACKGROUND: This retrospective pooled analysis assessed the effect of age on the efficacy and safety of trabectedin in young and elderly patients with recurrent advanced soft tissue sarcoma (STS). METHODS: Data from 350 adults with STS treated in five phase II trials with trabectedin were divided in the younger (<60 years; n=267) and the older cohort (≥60 years; n=83). RESULTS: The response rate did not differ with age (younger: 10.1% vs elderly 9.6%). No significant differences were found in median progression-free survival (PFS) in younger (2.5 months) and older (3.7 months) cohort with a comparable PFS rates at 3 (45.1% vs 55.1%) and 6 months (29.5% vs 36.4%). Similar median overall survival was observed in both cohorts (13.0 vs 14.0 months). Reversible neutropenia and aspartate aminotransferase/alanine aminotransferase elevation were the most common abnormalities. A higher incidence of grade 3/4 neutropenia (43.6% vs 60.2%) and fatigue (6.3% vs 14.4%) was observed in older patients. In 24 patients aged ≥70 years, no significant differences in efficacy or safety outcomes were found. CONCLUSION: This analysis demonstrated that trabectedin is a feasible treatment in young and elderly patients with STS, with meaningful clinical benefits and an acceptable safety profile, essential in palliative treatment of elderly patients.

Clinical outcomes and safety with trabectedin therapy in patients with advanced soft tissue sarcomas following failure of prior chemotherapy: results of a worldwide expanded access program study.

BACKGROUND: This expanded access program (EAP) was designed to provide trabectedin access for patients with incurable soft tissue sarcoma (STS) following progression of disease with standard therapy. The outcomes of trial participants accrued over approximately 5 years are reported. PATIENTS AND METHODS: Adult patients with advanced STS of multiple histologies, including leiomyosarcoma and liposarcoma (L-sarcomas), following relapse or disease progression following standard-of-care chemotherapy, were enrolled. Trabectedin treatment cycles (1.5 mg/m(2), intravenously over 24 h) were repeated q21 days. Objective response, overall survival (OS), and safety were evaluated. RESULTS: Of 1895 patients enrolled, 807 (43%) had evaluable objective response data, with stable disease reported in 343 (43%) as best response. L-sarcoma patients exhibited longer, OS compared with other histologies [16.2 months (95% confidence interval (CI) 14.1-19.5) versus 8.4 months (95% CI 7.1-10.7)], and a slightly higher objective response rate [6.9% (95% CI 4.8-9.6) versus 4.0% (95% CI 2.1-6.8)]. The median treatment duration was 70 days representing a median of three treatment cycles; 30% of patients received ≥ 6 cycles. Safety and tolerability in this EAP were consistent with prior clinical trial data. CONCLUSION: Results of this EAP are consistent with previous reports of trabectedin, demonstrating disease control despite a low incidence of objective responses in advanced STS patients after failure of standard chemotherapy. CLINICALTRIALS.GOV: NCT00210665.

Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development.

During the 3-h developmental stage 14 in the chick, intercellular transfer of iontophoresed fluorescent dyes becomes less sensitive to the lowering of intracellular pH by either CO2 or acetate ions. Up to developmental state 14, dye transfer between lens cells is reversibly blocked by exposure to 50% CO2. Beyond stage 14, dye transfer between these cells is no longer reversibly blocked by elevated pCO2. Electronic coupling is present throughout lens development and is not reversibly blocked by high pCO2 at any stage. The gap junctions joining the lens cells show morphological changes at developmental stage 14. Up to stage 14, all gap junctions observed between chick lens cells have connexon assemblies that appear condensed or crystalline following routine freeze-fracture microscopy. Beyond stage 14, chick lens cells express gap junctions with both the condensed assemblies and the dispersed assemblies characteristic of adult lens gap-junction structure.

Functional properties and developmental regulation of nicotinic acetylcholine receptors on embryonic chicken sympathetic neurons.

Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. Later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity.

Substance P modulates single-channel properties of neuronal nicotinic acetylcholine receptors.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.

The Ets-related transcription factor PU.1 immortalizes erythroblasts.

In vivo studies of Friend virus erythroleukemia have implied that proviral integrations adjacent to the gene for the Ets-related transcription factor PU.1 may inhibit the commitment of erythroblasts to differentiate and cause their capability for indefinite transplantation (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 62:4129-4135, 1988; R. Paul, S. Schuetze, S. L. Kozak, C. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test this hypothesis, we ligated PU.1 cDNA into a retroviral vector and studied its effects on cultured cells. Infection of fibroblasts with PU.1-encoding retrovirus resulted in PU.1 synthesis followed by nuclear pyknosis, cell rounding, and degeneration. In contrast, in long-term bone marrow cultures, erythroblasts were efficiently and rapidly immortalized. The resulting cell lines were polyclonal populations that contained PU.1, were morphologically blast-like, required erythropoietin and bone marrow stromal cells for survival and proliferation, and spontaneously differentiated at low frequency to synthesize hemoglobin. After 9 months in culture, erythroblasts became stroma independent, and they then grew as clonal cell lines. We conclude that PU.1 perturbs the pathway(s) that controls potential for indefinite proliferation and that it can be used to generate permanent erythroblast cell lines.

Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells.

Both viral and cellular genes have been directly implicated in pathogenesis of Friend viral erythroleukemia. The virus-encoded gp55 glycoprotein binds to erythropoietin receptors to cause mitogenesis and differentiation of erythroblasts. However, if the provirus integrates adjacent to the gene for the PU.1 transcription factor, the cell loses its commitment to terminally differentiate and becomes immortal, as indicated by its transplantability and by its potential for indefinite growth in culture (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 63:4434-4437, 1989; R. Paul, S. Schuetze, S. L. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test the implications of these results, we produced polyclonal antiserum to bacterially synthesized PU.1, and we used it to analyze PU.1 expression throughout leukemic progression and during chemically induced differentiation of Friend erythroleukemia (F-MEL) cell lines. This antiserum identified three electrophoretically distinct PU.1 components in extracts of F-MEL cells and demonstrated their nuclear localization. Although PU.1 proteins are abundant in F-MEL cells, they are absent or present in only trace amounts in normal erythroblasts or in differentiating erythroblasts from the preleukemic stage of Friend disease. Furthermore, chemicals (dimethyl sulfoxide or N,N'-hexamethylenebisacetamide) that overcome the blocked differentiation of F-MEL cells induce rapid declines of PU.1 mRNA and PU.1 proteins. The elimination of PU.1 proteins coincides with recommitment to the program of erythroid differentiation and with loss of immortality. These results support the hypothesis that PU.1 interferes with the commitment of erythroblasts to differentiate and that chemicals that reduce PU.1 expression reinstate the erythropoietic program.

Acquired protein S deficiency with multiple thrombotic complications after orthotopic liver transplant.

BACKGROUND: Orthotopic liver transplantation (OLT) is frequently complicated by thrombotic events that may threaten the viability of the allograft and severely compromise the overall outcome in these patients. Although multiple prothrombotic pathogenic mechanisms may be involved, a role for inadequate natural anticoagulant levels in the early postoperative period has been postulated. METHODS: We describe a case of a woman who suffered multiple thrombotic complications after a second OLT. Prospective assays of procoagulant and natural anticoagulant factor levels, in addition to screening tests for a variety of inherited and acquired hypercoagulable states, were carried out. RESULTS: Serial studies confirmed an acquired, isolated deficiency of Protein S associated with the second transplanted liver. Protein S levels were normal after the patient's first and third OLTs. There was no laboratory evidence of other underlying prothrombotic conditions. CONCLUSIONS: This unusual case of acquired Protein S deficiency demonstrates that the hypercoagulable phenotype may develop in the recipient of a liver from a heterozygous deficient donor. Furthermore, isolated low Protein S may be causally associated with hepatic artery thrombosis after OLT.

The acetylcholine channel open time in chick muscle is not decreased following innervation.

1. The mean channel open time (tau) of ACh receptors was measured in chick muscles at various stages of development. Tau was estimated by analysing ACh induced current fluctuations recorded extracellularly from small (ca. 20 micrometers2) membrane patches. 2. At random sites on uninnervated, embryonic chick muscle fibres in vitro, tau was relatively long--4 msec at 23 degrees C. 3. Estimates of tau at synaptic sites on embryonic myotubes innervated in vitro were identical to estimates at extrasynaptic sites on the same fibres. Both were comparable to estimates on uninnervated myotubes. 4. Synaptic currents at cultured junctions decayed slowly as simple exponentials. The decay time constants were never shorter than the mean channel open time measured by fluctuation analysis. 5. In anterior latissimus dorsi and intercostal muscle fibres of 4- to 18-week posthatched chicks, fluctuation analysis and synaptic current decays indicate that the channel open time of mature chick endplate receptors is as long as that of embryonic synaptic receptors in vitro. Apparently, tau remains prolonged throughout the maturation of chick neuromuscular junctions.

Development of rat soleus endplate membrane following denervation at birth.

Rat soleus endplates develop some of their characteristic features before birth and others after birth. Specializations appearing before birth include a localized cluster of acetylcholine receptors (AChRs), an accumulation of acetylcholinesterase (AChE) in the synaptic basal lamina, and a cluster of nuclei near the endplate membrane. In contrast, postsynaptic membrane folds are elaborated during the first three weeks after birth. We denervated soleus muscles on postnatal day 1, before folds had appeared, and followed the subsequent development of endplate regions with light and electron microscopy. We found that the denervated endplates initiated fold formation on schedule and maintained their accumulations of AChRs, AChE, and endplate nuclei. However, the endplates stopped fold formation prematurely and eventually lost their rudimentary folds. At about the same time, the junctional AChR clusters were joined by ectopic patches of AChRs. The former endplate regions also became unusually elongated, possibly as a consequence of the lack of membrane folds. Apparently, endplate membranes have only a limited capacity for further development in the absence of both the nerve and muscle activity.

Apparent acetylcholine receptor channel conversion at individual rat soleus end-plates in vitro.

Miniature end-plate currents (m.e.p.c.s.) were recorded extracellularly from individual fibres in neonatal rat soleus muscles for 2-24 h. In agreement with previous studies, the decay phases of m.e.p.c.s at many end-plates were doubly exponential with time constants of approximately 6 ms and approximately 1.5 ms at 21 degrees C. Earlier studies have shown that doubly exponential decays are due to the combined action of embryonic-type acetylcholine (ACh) receptors (AChRs) with long channel open times and adult-type AChRs with brief open times. When individual end-plates with doubly exponential m.e.p.c.s were studied for several hours or more, the relative size of the slow decay component frequently decreased with time. There was no evidence for a corresponding decrease in total m.e.p.c. amplitude. The time constants of the fast and slow components did not change. M.e.p.c. decays were stable at end-plates that were either very mature (small slow decay component) or very immature (small fast decay component). In these cases, the decay phases were virtually singly exponential and the time constant did not change. These data indicate that at end-plates with a mixture of adult-type and embryonic-type channels, the fraction of adult-type AChRs increases with time. This is similar to what occurs at end-plates developing in vivo. The results of ACh noise analysis experiments support this interpretation.

Kinetic differences between embryonic- and adult-type acetylcholine receptors in rat myotubes.

1. The burst structures of embryonic-type (low-gamma) and adult-type (high-gamma) nicotinic acetylcholine (ACh) receptors (AChRs) in rat myotubes were investigated with the patch clamp technique. The channels were activated with the agonists ACh and suberyldicholine (SubCh). 2. With either agonist, the distribution of burst durations showed two exponential components for both channel types: a 'long' component that corresponds to bursts of one or more openings and a 'brief' component that includes short, isolated openings. 3. For low-gamma channels, the percentage of all openings associated with the brief component decreased from approximately 40% at 10-100 nM-ACh to less than 10% at 10-100 microM-ACh. 4. Both high-gamma and low-gamma long bursts were interrupted by brief (30-90 microseconds) closures and longer (approximately 1 ms) transitions to a partially open (subconductance) state. The duration of brief closures and partial openings was relatively independent of the agonist, but their frequency within low-gamma bursts was 3-fold higher with SubCh than with ACh. 5. Brief closures are interpreted as transitions to a closed, doubly liganded state from which the channel can reopen. This model predicts that the channel opening rate is greater than 10,000 s-1 for both channel types at room temperature. 6. Estimates of the channel opening rate inferred from the rising phase of miniature end-plate currents recorded from rat soleus fibres are consistent with this interpretation. 7. Both high-gamma and low-gamma channels apparently operate via similar gating mechanisms. Differences in their gating behaviour can be explained in terms of faster kinetic rate constants for high-gamma channels.