Veliparib monotherapy following carboplatin/paclitaxel plus veliparib combination therapy in patients with germline BRCA-associated advanced breast cancer: results of exploratory analyses from the phase III BROCADE3 trial.

BACKGROUND: In the BROCADE3 trial, addition of the poly(ADP-ribose) polymerase inhibitor, veliparib, to carboplatin/paclitaxel improved progression-free survival (PFS) (hazard ratio 0.71, 95% confidence interval 0.57-0.88; P = 0.002) in patients with advanced human epidermal growth factor receptor 2-negative, germline BRCA1/2-mutated breast cancer. A subset of patients discontinued both carboplatin and paclitaxel before progression and continued on veliparib/placebo maintenance monotherapy until progression. Analyses in this patient subgroup are reported. PATIENTS AND METHODS: Patients were randomized 2 : 1 to veliparib plus carboplatin/paclitaxel or placebo plus carboplatin/paclitaxel. Veliparib (120 mg twice daily) or placebo was given on days -2 to 5, carboplatin (area under the curve 6 mg/ml) on day 1, and paclitaxel (80 mg/m2) on days 1, 8, and 15 of 21-day cycles. Patients who discontinued both carboplatin and paclitaxel before progression received blinded study drug monotherapy at an increased dose of 300-400 mg twice daily continuously. PFS was the primary endpoint. Exploratory analyses were carried out in the subgroup of patients who received blinded study drug as monotherapy. A time-varying Cox model including data from all patients was also used to evaluate treatment effect in the combination and monotherapy phases. RESULTS: A total of 136 of 337 patients randomized to veliparib plus carboplatin/paclitaxel and 58/172 patients randomized to placebo plus carboplatin/paclitaxel discontinued both carboplatin and paclitaxel before progression and continued on blinded veliparib or placebo monotherapy. In this blinded monotherapy subgroup, investigator-assessed median PFS from randomization was 25.7 months with veliparib versus 14.6 months with placebo. Hazard ratios from a time-varying Cox model favored veliparib during both combination therapy and monotherapy. Any-grade adverse events occurring in the monotherapy phase were primarily gastrointestinal. The most common grade ≥3 adverse events were neutropenia and anemia (4% each with veliparib; 5% and 2%, respectively, with placebo). CONCLUSIONS: Veliparib maintenance monotherapy had a tolerable safety profile and may extend PFS following combination chemotherapy.

Dynamic molecular analysis and clinical correlates of tumor evolution within a phase II trial of panitumumab-based therapy in metastatic colorectal cancer.

Background: Mutations in rat sarcoma (RAS) genes may be a mechanism of secondary resistance in epidermal growth factor receptor inhibitor-treated patients. Tumor-tissue biopsy testing has been the standard for evaluating mutational status; however, plasma testing of cell-free DNA has been shown to be a more sensitive method for detecting clonal evolution. Materials and methods: Archival pre- and post-treatment tumor biopsy samples from a phase II study of panitumumab in combination with irinotecan in patients with metastatic colorectal cancer (mCRC) that also collected plasma samples before, during, and after treatment were analyzed for emergence of mutations during/post-treatment by next-generation sequencing and BEAMing. Results: The rate of emergence of tumor tissue RAS mutations was 9.5% by next-generation sequencing (n = 21) and 6.3% by BEAMing (n = 16). Plasma testing of cell-free DNA by BEAMing revealed a mutant RAS emergence rate of 36.7% (n = 39). Exploratory outcomes analysis of plasma samples indicated that patients who had emergent RAS mutations at progression had similar median progression-free survival to those patients who remained wild-type at progression. Serial analysis of plasma samples showed that the first detected emergence of RAS mutations preceded progression by a median of 3.6 months (range, -0.3 to 7.5 months) and that there did not appear to be a mutant RAS allele frequency threshold that could predict near-term outcomes. Conclusions: This first prospective analysis in mCRC showed that serial plasma biopsies are more inclusive than tissue biopsies for evaluating global tumor heterogeneity; however, the clinical utility of plasma testing in mCRC remains to be further explored. ClinicalTrials.gov Identifier: NCT00891930.

Health-related quality of life in women with recurrent ovarian cancer receiving paclitaxel plus trebananib or placebo (TRINOVA-1).

BACKGROUND: To evaluate the influence of treatment on health-related quality of life (HRQoL) in 919 women with recurrent ovarian cancer enrolled in the TRINOVA-1 study, a randomized, placebo-controlled phase III study that demonstrated that trebananib 15 mg/kg QW plus weekly paclitaxel significantly improved progression-free survival (PFS) compared with placebo plus weekly paclitaxel (7.2 versus 5.4 months; hazard ratio, 0.66; 95% confidence interval 0.57-0.77; P < 0.001). PATIENTS AND METHODS: HRQoL was assessed with the Functional Assessment of Cancer Therapy-Ovary [FACT-O; comprising FACT-G and the ovarian cancer-specific subscale (OCS)] and EuroQOL EQ-5D instruments before treatment on day 1 of weeks 1, 5, 9, 13, 17, and every 8 weeks thereafter and at the safety follow-up visit. A pattern-mixture model was used to evaluate the influence of patient dropout on FACT-O and OCS scores over time. RESULTS: Of 919 randomized patients, 834 (91%) had a baseline and ≥1 post-baseline HRQoL assessment. At baseline, scores for all instruments were similar for both arms. At 25 weeks, mean ± SD changes from baseline were negligible, with mean ± SD changes typically <1 unit from baseline: -2.4 ± 16.6 in the trebananib arm and -1.6 ± 15.2 in the placebo arm for FACT-O, -0.71 ± 5.5 in the trebananib arm and -0.86 ± 4.9 in the placebo arm for OCS, and -0.02 ± 0.22 in the trebananib arm and 0.02 ± 0.19 in the placebo arm for EQ-5D. Distribution of scores was similar between treatment arms at baseline and over the course of the study. In pattern-mixture models, there was no evidence that patient dropout affected differences in mean FACT-O or OCS scores. Edema had limited effect on either FACT-O or OCS scores in patients with grade ≥2 edema or those with grade 1 or no edema. CONCLUSIONS: Our results demonstrate that the improvement in PFS among patients in the trebananib arm in the TRINOVA-1 study was achieved without compromising HRQoL. CLINICALTRIALSGOV IDENTIFIER: NCT01204749.

A phase 3 randomized, double-blind, placebo-controlled trial of ganitumab or placebo in combination with gemcitabine as first-line therapy for metastatic adenocarcinoma of the pancreas: the GAMMA trial.

BACKGROUND: This double-blind, phase 3 study assessed the efficacy and safety of ganitumab combined with gemcitabine as first-line treatment of metastatic pancreatic cancer. PATIENTS AND METHODS: Patients with previously untreated metastatic pancreatic adenocarcinoma were randomly assigned 2 : 2 : 1 to receive intravenous gemcitabine 1000 mg/m(2) (days 1, 8, and 15 of each 28-day cycle) plus placebo, ganitumab 12 mg/kg, or ganitumab 20 mg/kg (days 1 and 15 of each cycle). The primary end point was overall survival (OS). Secondary end points included progression-free survival (PFS), safety, and efficacy by levels of circulating biomarkers. RESULTS: Overall, 322 patients were randomly assigned to placebo, 318 to ganitumab 12 mg/kg, and 160 to ganitumab 20 mg/kg. The study was stopped based on results from a preplanned futility analysis; the final results are reported. Median OS was 7.2 months [95% confidence interval (CI), 6.3-8.2] in the placebo arm, 7.0 months (95% CI, 6.2-8.5) in the ganitumab 12-mg/kg arm [hazard ratio (HR), 1.00; 95% CI, 0.82-1.21; P = 0.494], and 7.1 months (95% CI, 6.4-8.5) in the ganitumab 20-mg/kg arm (HR, 0.97; 95% CI, 0.76-1.23; P = 0.397). Median PFS was 3.7, 3.6 (HR, 1.00; 95% CI, 0.84-1.20; P = 0.520), and 3.7 months (HR, 0.97; 95% CI, 0.77-1.22; P = 0.403), respectively. No unexpected toxicity was observed with ganitumab plus gemcitabine. The circulating biomarkers assessed [insulin-like growth factor-1 (IGF-1), IGF-binding protein-2, and -3] were not associated with a treatment effect on OS or PFS by ganitumab. CONCLUSION: Ganitumab combined with gemcitabine had manageable toxicity but did not improve OS, compared with gemcitabine alone in unselected patients with metastatic pancreatic cancer. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT01231347.

The significance of activation markers on CD8 lymphocytes in human immunodeficiency syndrome: staging and prognostic value.

The objective of this prospective cohort study was to evaluate the expression of activation markers on CD8 lymphocytes at various clinical stages of HIV infection and to determine the value of these markers in identifying patients likely to have rapidly progressive disease. One hundred and three HIV+ patients, divided into four disease stages, and 34 seronegative controls were evaluated at study entry using flow cytometric immunophenotyping. The HIV patients were followed clinically for disease progression during the following 2 years. CD8 cell numbers and percentage of lymphocytes are increased after HIV infection. Expression of the CD38, HLA-DR and CD57 markers on CD8 cells was significantly increased in asymptomatic HIV-infected patients when compared with controls, as was the CD8 cell population which did not coexpress Leu-8. These activation markers were observed to be further increased in patient groups with more clinically advanced infection. The percentage of CD38 on CD8 cells emerged not only as a discriminator of disease severity, but was a strong predictor of progression in asymptomatic, lymphadenopathy and ARC patients. Given the utility of activation markers on CD8 lymphocytes in staging disease and predicting clinical outcome, the measurement of these parameters should be considered in the monitoring and management of HIV patients.

Expression of activation antigens, HLA-DR and CD38, on CD8 lymphocytes during HIV-1 infection.

OBJECTIVE: To study the expression of the activation markers human leukocyte antigen (HLA)-DR and CD38 antigen on CD8+ T-lymphocytes in HIV-infected subjects and HIV-negative controls. DESIGN: Two- and three-colour flow-cytometric analysis. METHODS: Fresh peripheral venous blood was obtained from 16 HIV-infected subjects, representing four different stages of HIV disease, and from six HIV-negative controls. Three-colour lymphocyte immunophenotyping was performed using peridinyl chlorophyll-A protein (PerCP)-conjugated anti-CD8 monoclonal antibody (MAb) in combination with anti-HLA-DR (phycoerythrin) and anti-CD38 (fluorescein isothiocyanate) MAb. RESULTS: The relative percentage of the lymphocyte populations thus defined differed between HIV-negative and HIV-positive subjects and between HIV-infected subjects at different clinical stages of disease. Simultaneous expression of HLA-DR and CD38 within the CD8 T-lymphocyte compartment increased from 8% in controls to 49% in asymptomatic HIV-infected subjects (P less than 0.005). Symptomatic patients differed from asymptomatic seropositives by a further increase in the HLA-DR+ CD38+ CD8 subset. In AIDS patients, the HLA-DR+ CD38- CD8 subset decreased (P less than 0.05) and the HLA-DR- CD38+ CD8 subset increased (P less than 0.05), compared with the other HIV disease stage patients. CONCLUSION: There is a stage-associated pattern of HLA-DR and CD38 expression on CD8 T-lymphocytes during HIV infection; specific phenotypic patterns may have functional correlates in the host response to the virus.

Improved sensitivity and resolution in the flow cytometric DNA analysis of human solid tumor specimens. Use of in vitro fine-needle aspiration and uniform staining reagents.

Twenty-five unselected fresh colon or breast tumors were studied to identify specific components of sample preparation, sample staining, and flow cytometer operation affecting the sensitivity of DNA stemline analysis. Solid tumors were disaggregated using both a published method for mechanical/enzymatic whole cell (M/EWC) isolation and a fine-needle aspiration (FNA) technique. Staining FNA samples with CycleTEST propidium iodide reagents demonstrated improved sensitivity in the recognition of near diploid and near tetraploid aneuploid populations: 9 of 20 resolvable aneuploid DNA stemlines identified in FNA suspensions were not detected or clearly resolved in M/EWC preparations. These results suggest that previously reported discordances between flow and static image cytometry in the recognition of near tetraploid DNA stemlines may be related to inherent limitations of M/EWC. The in vitro FNA technique, when compared to M/EWC, may yield increased sensitivity and precision in clinical DNA stemline analysis when using fresh, unfixed, solid tumor specimens.

Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry.

It is possible accurately to distinguish lymphocytes from other leukocyte populations in peripheral blood using the combination of fluorescence associated with CD45/CD14 and forward and orthogonal light scatter. By identifying the cell population of interest based on immunofluorescence, a light scattering window can then be drawn to include all (greater than or equal to 98%) of the lymphocytes. In this manner, maximal recovery of the lymphocytes within a sample can be consistently obtained. The combination of light scattering and immunofluorescence can also be used to define the purity of the gate. The identification of nonlymphocytes within the light scattering gate can then be used to establish an accurate denominator for the percent lymphocytes stained. Once the optimal data acquisition gate has been established and characterized, it is possible to correct subsequent analyses with that particular sample since the reactivity of monoclonal antibodies on monocytes and granulocytes can be accounted for once the nonlymphocytes have been identified as being within the acquisition gate.

Genetic and serological analysis of the expression of crossreactive idiotypic determinants on anti-p-azobenzenarsonate antibodies and p-azobenzenarsonate-specific suppressor T cell factors.

Data are reported on the genetic control and structure of antigen-specific suppressive molecules obtained from azobenzenearsonate (ABA)-specific suppressor T cells (Ts). Ts-derived suppressor factors (TsFs) were previously shown to bear determinants encoded by genes of the I-J subregion of the H-3 major histocompatibility complex and structures encoded by VH (variable region of heavy chains) genes linked to the Igh locus. To further characterize TsF we have made use of a K light (L) chain variable region (VK) genetic marker linked to the locus governing expression of a crossreactive idiotype on anti-ABA antibodies. The experiments evaluated the possible contribution to TsF of structures under control of the VK locus. TsF was prepared in strains of mice with known Igh and VK genes. Mice carrying Igh-1e genes produced TsF that was retained by an immunoadsorbent containing bound anti-idiotype antibodies; the genetic constitution at the VK locus was not relevant. By using a panel of anti-idiotypic antibodies prepared against the antigen-binding site of antibody carrying the idiotype, against determinants outside the site or against VH structures, we determined that idiotypic structures on the TsF are associated with V-region elements associated with the H chain and not with the binding site formed by a VH-BL combination.

Antigen- and receptor-driven regulatory mechanisms. I. Induction of suppressor T cells with anti-idiotypic antibodies.

Delayed-type hypersensitivity (DTH) to the azobenzenearsonate (ABA) hapten can be readily induced in A/J mice injecting ABA-coupled syngeneic spleen cells subcutaneously. To further characterize this T-cell-dependent immunological phenomenon, the effect of passively administered anti-cross-reactive idiotype common to anti-ABA antibodies of A/J mice (CRI) antibodies on the development of ABA-specific DTH was investigated. Animals given daily injections (of minute amounts) of anti-CRI antibodies subsequent to immunization with ABA-coupled cells show significant reduction of ABA specific responses. This inhibition is antigen specific and requires the intact immunoglobulin molecule, as F(ab')2 treatments were ineffective in suppressing the reaction. Investigations of the mechanism of the anti-CRI-induced suppression of ABA DTH revealed that the observed suppression is a result of the activation of suppressor cells. Spleen cells taken from animals which received anti-CRI antibodies were able to adoptively transfer suppression to naive recipients. This suppression was shown to be mediated by T cells, as anti-Thy1.2 plus complement completely abrogated the transfer of suppression. In addition, animals pretreated with low doses of cyclophosphamide were not suppressed by the administration of anti-CRI antibodies. The genetic restriction of anti-CRI-induced suppression was demonstrated. Antibodies to the major cross-reactive idiotype, (CRI) associated with anti-ABA antibodies in A/J mice were unable to suppress the development of DTH to ABA in BALB/c mice (H-2d, Igh-1a). Such antibodies were, however, fully active in suppressing ABA DTH in the allotype-congenic C.AL-20 strain which has an allotype (Igh-1d) similar to that of A/J (Igh-1e) on a BALB/c background, and which produces humoral antibodies with the CRI.

Antigen- and receptor-driven regulatory mechanisms. II. Induction of suppressor T cells with idiotype-coupled syngeneic spleen cells.

Anti-p-azobenzenearsonate (ABA) antibodies, coupled covalently to normal syngeneic spleen cells and then given intravenously to normal animals, were found to be potent tolerogens for delayed-type hypersensitivity (DTH) to ABA. The ability of the antibody-coupled cells to induce tolerance was determined to be a result of the cross-reactive idiotype (CRI+) fraction of the antibodies, because anti-ABA antibodies lacking the CRI+ components when coupled to spleen cells were unable to cause any significant inhibition. Furthermore, genetic analysis revealed that the ability of CRI-coupled cells to inhibit ABA-specific DTH is linked to Igh-1 heavy chain allotype, in as much animals which possess heavy chain allotypes similar to that of A/J were sensitive to this inhibition. Adoptive transfer experiments provided evidence that CRI-coupled cells induce suppressor cells, and spleen cells or thymocytes from animals received CRI-coupled cells were able to transfer suppression to naive recipients. In addition, treatment with anti-Thy1.2 serum plus complement completely abrogated their ability to transfer suppression. Thus, this active suppression is a T-cell-dependent phenomenon. In investigating the specificity of these suppressor T cells, it was found that they functioned in an antigen-specific manner and were unable to suppress the development of DTH to an unrelated hapten 2,4-dinitro-1-fluorobenzene.

Mechanisms of regulation of cell-mediated immunity. III. The characterization of azobenzenearsonate-specific suppressor T-cell-derived-suppressor factors.

Delayed type hypersensitivity to the hapten azobenzenearsonate (ABA) can be induced and suppressed by the administration of hapten-coupled syngeneic spleen cells by the appropriate route. Suppressor T cells stimulated by the intravenous administration of ABA-coupled spleen cells have been shown to produce a discrete subcellular factor(s) which is capable of suppressing delayed type hypersensitivity to azobenzenearsonate in the mouse. Such suppressor factors may be produced by the mechanical disruption of suppressor cells or by placing such suppressor cells in culture for 24 h. The suppressor factor(s) (SF) derived from ABA-specific suppressor cells exhibit biological specificity for the suppression of ABA delayed type hypersensitivity (DTH), but not trinitro-phenyl DTH, as well as the capacity to bind to ABA immunoadsorbents. Passage of suppressor factor(s) over reverse immunoadsorbents utilizing a rabbit anti-mouse F(ab')2 antiserum demonstrated that the antigen-specific T-cell derived SF does not bear conventional immunoglobulin markers. The suppressor factor(s) are not immunoglobulin molecules was further demonstrated by the inability of anti-ABA antibodies to suppress ABA DTH. Gel filtration of ABA suppressor factor(s) showed that the majority of the suppressive activity was present in a fraction with molecular weight ranging between 6.8 x 10(4) and 3.3 x 10(4) daltons. We also analyzed for the presence of determinants encoded by the H-2 major histocompatibility complex (MHC) and found that immunoadsorbents prepared utilizing antisera capable of interacting with gene products of the whole or selected gene regions of H-2 MHC, i.e., B10.D2 anti-B10.A and B10 anti-B10.A immunoadsorbents, retained the suppressive activity of ABA-SF. Elution of such columns with glycine HCl buffers (pH 2.8) permitted recovery of specific suppressive activity. Taken collectively such data supports the notion that suppressor T-cell-derived ABA suppressor factors have antigen-binding specificity as well as determinants controlled by the K end of the H-2 MHC. The distribution of strains capable of making SF has also been analyzed. The relationship of the antigen-binding specificity to VH gene products is discussed in this and the companion paper.

Mechanisms of regulation of cell-mediated immunity. IV. Azobenzenearsonate-specific suppressor factor(s) bear cross-reactive idiotypic determinants the expression of which is linked to the heavy-chain allotype linkage group of genes.

T-cell derived suppressor factor(s) (SF) specific for azobenzenearsonate (ABA) were prepared by the mechanical disruption of suppressor cells. Such suppressor factors were adsorbed to and recovered from immunoadsorbents prepared from the F(ab')2 fragments of rabbit immunoglobulin directed against the cross-reactive idiotype of A/J anti-ABA antibodies. These ABA-suppressor factors were not retained on Sepharose 4B immunoadsorbent columns which had been coupled with F(ab')2 fragments or normal rabbit immunoglobulins prepared from prebleeds of rabbits used to make anti-idiotypic antiserum. The specificity of the F(ab')2 rabbit anti-idiotypic serum was established by direct idiotypic-binding assays and by affinity purification over an immunoadsorbent consisting of CRI+ anti-ABA immunoglobulin from A/J mice. ABA-suppressor factors were shown to be specifically absorbed and eluted from F(ab')2 anti-idiotypic columns. Futhermore, the eluted suppressor factor can be specifically reabsorbed and recovered from a second anti-idiotypic immunoadsorbent. The concordance between antigen-binding specificity and the presence of idiotypic determinants was demonstrated by adsorbing ABA SF to antigen columns and then fractionating the ABA-specific factor on anti-idiotypic immunoadsorbents. ABA-suppressor factors were shown to be specifically retained on immunoadsorbents directed against major histocompatibility complex (MHC) determinants. Factor eluted from anti-MHC columns could then be specifically adsorbed to anti-idiotypic immunoadsorbents. This suggests that the same molecular complex that is recognized by the H-2 alloantiserum is specifically adsorbed to an anti-idiotypic immunoadsorbent. Genetic analysis of the expression of CRI+ suppressor factor was performed using the C.AL-20 mouse strain which has the AL/N allotype and produces CRI+ anti-ABA immunoglobulins. The implication of these findings to the nature of T-cell-derived regulatory molecules is discussed.