A phase 2, randomized, double-blind, placebo- controlled study of chemo-immunotherapy combination using motolimod with pegylated liposomal doxorubicin in recurrent or persistent ovarian cancer: a Gynecologic Oncology Group partners study.

BACKGROUND: A phase 2, randomized, placebo-controlled trial was conducted in women with recurrent epithelial ovarian carcinoma to evaluate the efficacy and safety of motolimod-a Toll-like receptor 8 (TLR8) agonist that stimulates robust innate immune responses-combined with pegylated liposomal doxorubicin (PLD), a chemotherapeutic that induces immunogenic cell death. PATIENTS AND METHODS: Women with ovarian, fallopian tube, or primary peritoneal carcinoma were randomized 1 : 1 to receive PLD in combination with blinded motolimod or placebo. Randomization was stratified by platinum-free interval (≤6 versus >6-12 months) and Gynecologic Oncology Group (GOG) performance status (0 versus 1). Treatment cycles were repeated every 28 days until disease progression. RESULTS: The addition of motolimod to PLD did not significantly improve overall survival (OS; log rank one-sided P = 0.923, HR = 1.22) or progression-free survival (PFS; log rank one-sided P = 0.943, HR = 1.21). The combination was well tolerated, with no synergistic or unexpected serious toxicity. Most patients experienced adverse events of fatigue, anemia, nausea, decreased white blood cells, and constipation. In pre-specified subgroup analyses, motolimod-treated patients who experienced injection site reactions (ISR) had a lower risk of death compared with those who did not experience ISR. Additionally, pre-treatment in vitro responses of immune biomarkers to TLR8 stimulation predicted OS outcomes in patients receiving motolimod on study. Immune score (tumor infiltrating lymphocytes; TIL), TLR8 single-nucleotide polymorphisms, mutational status in BRCA and other DNA repair genes, and autoantibody biomarkers did not correlate with OS or PFS. CONCLUSIONS: The addition of motolimod to PLD did not improve clinical outcomes compared with placebo. However, subset analyses identified statistically significant differences in the OS of motolimod-treated patients on the basis of ISR and in vitro immune responses. Collectively, these data may provide important clues for identifying patients for treatment with immunomodulatory agents in novel combinations and/or delivery approaches. TRIAL REGISTRATION: Clinicaltrials.gov, NCT 01666444.

A randomized phase II study of the telomerase inhibitor imetelstat as maintenance therapy for advanced non-small-cell lung cancer.

BACKGROUND: Continuation or 'switch' maintenance therapy is commonly used in patients with advancd non-small-cell lung cancer (NSCLC). Here, we evaluated the efficacy of the telomerase inhibitor, imetelstat, as switch maintenance therapy in patients with advanced NSCLC. PATIENTS AND METHODS: The primary end point of this open-label, randomized phase II study was progression-free survival (PFS). Patients with non-progressive, advanced NSCLC after platinum-based doublet (first-line) chemotherapy (with or without bevacizumab), any histology, with Eastern Cooperative Oncology Group performance status 0-1 were eligible. Randomization was 2 : 1 in favor of imetelstat, administered at 9.4 mg/kg on days 1 and 8 of a 21-day cycle, or observation. Telomere length (TL) biomarker exploratory analysis was carried out in tumor tissue by quantitative PCR (qPCR) and telomerase fluorescence in situ hybridization. RESULTS: Of 116 patients enrolled, 114 were evaluable. Grade 3/4 neutropenia and thrombocytopenia were more frequent with imetelstat. Median PFS was 2.8 and 2.6 months for imetelstat-treated versus control [hazard ratio (HR) = 0.844; 95% CI 0.54-1.31; P = 0.446]. Median survival time favored imetelstat (14.3 versus 11.5 months), although not significantly (HR = 0.68; 95% CI 0.41-1.12; P = 0.129). Exploratory analysis demonstrated a trend toward longer median PFS (HR = 0.43; 95% CI 0.14-1.3; P = 0.124) and overall survival (OS; HR = 0.41; 95% CI 0.11-1.46; P = 0.155) in imetelstat-treated patients with short TL, but no improvement in median PFS and OS in patients with long TL (HR = 0.86; 95% CI 0.39-1.88; and HR = 0.51; 95% CI 0.2-1.28; P = 0.145). CONCLUSIONS: Maintenance imetelstat failed to improve PFS in advanced NSCLC patients responding to first-line therapy. There was a trend toward a improvement in median PFS and OS in patients with short TL. Short TL as a predictive biomarker will require further investigation for the clinical development of imetelstat.

Chronic Helicobacter pylori infection induces an apoptosis-resistant phenotype associated with decreased expression of p27(kip1).

Helicobacter pylori infection is associated with the development of gastric cancer. In short-term coculture with AGS gastric cells, H. pylori inhibits cell cycle progression and induces dose-dependent apoptosis. Based on the concept that an imbalance between proliferation and apoptosis may contribute to the emergence of gastric cancer, we chronically exposed AGS cells to H. pylori as a model of chronic exposure in humans. The AGS derivatives selected by this process were stably resistant not only to H. pylori-induced apoptosis but also to apoptosis induced by other enteric bacteria and by several toxic agents including radiation and cancer chemotherapy. Like the parental AGS cells, the derivatives underwent G(1)/S-phase cell cycle inhibition in response to H. pylori. The AGS derivatives displayed a marked decrease in cellular levels of the cell cycle control protein p27(kip1). We found a similar decrease in epithelial cell p27(kip1) expression in gastric biopsy specimens from H. pylori-infected patients. These findings are consistent with observations that link decreases in the p27(kip1) level to increased susceptibility to cancer in mice with p27(kip1) deleted and to a poor prognosis of gastric cancer in humans. This is the first demonstration that bacterial infection can lead to apoptosis resistance and to cross-resistance to other inducers of apoptosis such as bacteria, chemotherapeutic agents, and radiation. The development of apoptosis resistance and downmodulation of p27(kip1) may contribute to the increased risk for gastric cancer observed in humans chronically exposed to H. pylori.

Granulocyte-monocyte colony forming unit content of autologous bone marrow transplants in patients with haematological malignancy.

Cell viability and number of granulocyte-monocyte colony forming units (CFU-GM) were systematically assessed in 57 patients who had undergone transplantation of the autologous bone marrow for treatment of haematologic malignancies. Bone marrow cell cultivation in agarose with feeder layers appeared inferior to that performed in agarose with recombinant human granulocyte-monocyte colony stimulating factor and methylcellulose with phytohaemaglutinin leukocyte-conditioned medium. Since the transplant cells were frozen in liquid nitrogen between harvesting and reinfusion, the following samples were tested: buffy coat cells, buffy coat cells immediately after addition of dimethylsulphoxide, cell sample that had been frozen for 24 hours, and frozen transplant cells at the time of thawing and transplantation. Each procedural step decreased both cell viability and the number of CFU-GM, but since the lymphohaematologic recovery in all patients followed the pattern reported in the literature for high-quality transplants, we concluded that our transplants retained the necessary number of progenitor cells. It appears that the best strategy for dynamic assessment of the transplant quality would be to perform tests after every step of the transplant processing. Cell viability and number of progenitors per body weight in transplants were also found to be associated with probability of neutrophil reconstitution after bone marrow reinfusion.

Characterization and partial purification of the Croatian national standard Dermatophagoides pteronyssinus allergen extract.

Lyophilized Dermatophagoides pteronyssinus (Der p) allergen extract (AE) and partially purified Der p extract (PAE) were prepared and characterized. Partial purification of AE was performed by gel filtration on Sephadex G-100 and Sephacryl S-300. Crossed immunoelectrophoresis (CIE) disclosed the same precipitating lines in AE and PAE preparations. The relative potencies of AE and PAE were determined and compared with the WHO International Standard for Der p by the RAST inhibition method. The potencies were 6.5 x 10(5) IU and 1.5 x 10(6) IU, respectively. Biologic standardization by quantitative skin testing was performed with AE (20 selected patients) and PAE (12 patients). Median Ch was calculated by linear regression analysis (log-log model). One ampoule of AE contained 65,300 BU and 1 ml (vial) of PAE contained 166,000 BU. Der p AE could serve as a croatian national standard for further production of Der p allergenic extracts.

[Procollagen type 111 peptide levels in patients with bronchial asthma and COPD]. / Koncentracija prokolagen 111 peptida u bolesnika s bronhalnom astmom i KOPB.

The aim of these study was to evaluate if the inflammatory injury of parenchymal cells can lead to proliferation of fibroblasts and deposition of increased amounts of collagen, measured by the increased concentration of procollagen in blood, in patients with asthma and COPD. We evaluated 22 patients with asthma that has lasted more than 5 years, 18 patients with COPD and 20 healthy subjects. RIA-gnost method (Behring) was used to measure the procollagen peptide concentrations in blood. Our results showed that the concentration of procollagen peptide in blood samples from patients with asthma was 5.8 +/- 2.4, 4.9 +/- 1.8 in patients with COPD and 11.1 +/- 3.6 in healthy subjects. There was no significant difference between patients with asthma, COPD and healthy subjects (p less than 0.01). It can be concluded that there is no increased deposition of collagen in patients with long lasting asthma and COPD. Further studies of active collagen deposition in the early acute forms of these diseases are in progress.

[Treatment of neoplastic hematologic diseases with intensive radio-chemotherapy and transplantation of cryopreserved autologous bone marrow]. / Lijecenje neoplastickih hematoloskih bolesti intenzivnom radiokemoterapijom i transplantacijom krioprezervirane autologne kostane srzi.

Autologous bone marrow transplantation (ABMT) allows application of intensive myeloablative therapy aimed at eradication of neoplastic disease by facilitating haematopoietic reconstitution. Between March and June 1988, four patients (two with acute myelogenous leukaemia in first remission, one with acute lymphoblastic leukaemia in second remission, and one with Burkitt lymphoma, stage IV with CNS involvement in second remission) received this treatment. Methods of collecting, processing and freezing bone marrow as well as thawing and reinfusion of the marrow into patients after intensive chemoradiotherapy are described. Viability of bone marrow cells tested by the dye exclusion method after freezing and thawing process was 89, 88, 91 and 78%, respectively. CFU-GM recovery in culture, as a test of marrow stem cells clonogenicity was between 63,3 and 156,5%. Patients received between 1,7 and 3,0 x 10(8)/kg nucleated cells and 4,0 to 7,6 x 10(4)/kg CFU-GM, respectively. In all four patients stable haematopoietic reconstitution was achieved. The bone marrow function was evident mainly at 11th day after marrow reinfusion. Leukocyte count reached 1,0 x 10(0)/L in 11 to 15 days, and granulocyte count raised more than 0,5 x 10(9)/L in 19 to 37 days after transplantation. Platelet recovery was prolonged with the minimum of 29 days and maximum of more than 60 days to reach 20 x 10(9)/L. Side effects caused by the intensive radiochemotherapy were moderate. Bacterial, fungal and viral infections in early posttransplant period were successfully treated. All patients have survived and left the hospital 63, 54, 36 and 65 days after ABMT, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Accuracy of routine flow-cytometric bitmap selection for three leukocyte populations.

A double-blind study was performed with peripheral blood of 41 human subjects to check the accuracy of determination of lymphocyte, monocyte, and granulocyte windows with which every flow cytometric analysis of leukocyte markers starts. White blood cell suspensions were prepared according to the whole blood method and analyzed on an EPICS-C flow cytometer using the two-parameter 90 degrees light scatter vs. forward angle light scatter (granularity vs. cell size) data distribution. Windows (bitmaps) for lymphocytes, monocytes, and granulocytes were drawn and numbers of cells determined in each. The proportions of lymphocytes, monocytes, and granulocytes were calculated in relation to total cell number, counted and in relation to the sum of cells in three bitmaps, and then compared with proportions determined by microscopic whole blood cell (WBC) differential and a WBC differential determined in an automated hematology analyzer. Average proportions of lymphocytes obtained by the flow cytometer were significantly lower than those obtained by either microscopic or automated differential, suggesting that some of the relevant cells were not included in the bitmaps. Granulocyte proportion related to total cell number was lower and that related to bitmap cell number higher than that obtained by microscopic and automatic differentials, suggesting that nongranulocytic cells were included in the granulocyte bitmaps. Proportions of lymphocytes and granulocytes obtained by the flow cytometer correlated well with those obtained by both microscopic and automatic differential. In contrast, the proportions of monocytes showed a poor correlation, which is probably due to their low number and delicate position in the distribution, and which makes them difficult to delineate.

Lymphocyte subsets in normal human bone marrow harvested for routine clinical transplantation.

Bone marrow and peripheral blood of 25 healthy bone marrow donors from our allogeneic bone marrow transplantation program were assessed for cell subsets bearing T11(CD2), T4(CD4), T8(CD8), B1(CD20) J5(CALLA, CD10), Mo1(CD11b), MY7(CD13). Mo2(CD14), MY9(CD33) and NKH-1 antigens. Bone marrow cell samples were taken for analysis at the start or at the end of the harvesting procedure of aspiration from the iliac crest. All samples were analysed on a flow cytometer at the lymphocyte window as obtained on the two-parameter (L90oLSxFALS) scatter diagram. There were no differences in the lymphocyte subset composition of bone marrow samples taken at the start or at the end of the harvesting procedure. In contrast to the majority of literature data, a high CD4/CD8 ratio was detected in bone marrow samples: it did not differ from that in the peripheral blood. The proportions of CD2 and CD4 T cell markers in the bone marrow correlated with those in the peripheral blood, thus further documenting a substantial bone marrow contamination with peripheral blood cells. A relatively large aspirate volume (4-5 ml) obtained from individual aspiration sites was identified as the only factor possibly accounting for the high-level contamination of bone marrow samples with peripheral blood. This conclusion was corroborated by low T cell proportions and low CD4/CD8 ratios found in the bone marrow washed from bone fragments and in bone marrow samples aspirated at first bone puncture in a volume of 1.0 ml. Taken together, these findings imply that less vigorous suction may decrease the number of T lymphocytes in bone marrow harvested for transplantation purposes.