EGFR pathway biomarkers in erlotinib-treated patients with advanced pancreatic cancer: translational results from the randomised, crossover phase 3 trial AIO-PK0104.

BACKGROUND: We aimed to identify molecular epidermal growth factor receptor (EGFR) tissue biomarkers in pancreatic cancer (PC) patients treated with the anti-EGFR agent erlotinib within the phase 3 randomised AIO-PK0104 study. METHODS: AIO-PK0104 was a multicenter trial comparing gemcitabine/erlotinib followed by capecitabine with capecitabine/erlotinib followed by gemcitabine in advanced PC; primary study end point was the time-to-treatment failure after first- and second-line therapy (TTF2). Translational analyses were performed for KRAS exon 2 mutations, EGFR expression, PTEN expression, the EGFR intron 1 and exon 13 R497K polymorphism (PM). Biomarker data were correlated with TTF, overall survival (OS) and skin rash. RESULTS: Archival tumour tissue was available from 208 (74%) of the randomised patients. The KRAS mutations were found in 70% (121 out of 173) of patients and exclusively occurred in codon 12. The EGFR overexpression was detected in 89 out of 181 patients (49%) by immunohistochemistry (IHC), and 77 out of 166 patients (46%) had an EGFR gene amplification by fluorescence in-situ hybridisation (FISH); 30 out of 171 patients (18%) had a loss of PTEN expression, which was associated with an inferior TTF1 (first-line therapy; HR 0.61, P=0.02) and TTF2 (HR 0.66, P=0.04). The KRAS wild-type status was associated with improved OS (HR 1.68, P=0.005); no significant OS correlation was found for EGFR-IHC (HR 0.96), EGFR-FISH (HR 1.22), PTEN-IHC (HR 0.77), intron 1 (HR 0.91) or exon 13 R497K PM (HR 0.83). None of the six biomarkers correlated with the occurrence of skin rash. CONCLUSION: The KRAS wild-type was associated with an improved OS in erlotinib-treated PC patients in this phase 3 study; it remains to be defined whether this association is prognostic or predictive.

Capecitabine plus oxaliplatin (CapOx) versus capecitabine plus gemcitabine (CapGem) versus gemcitabine plus oxaliplatin (mGemOx): final results of a multicenter randomized phase II trial in advanced pancreatic cancer.

BACKGROUND: To compare the efficacy and safety of three different chemotherapy doublets in the treatment of advanced pancreatic cancer (PC). PATIENTS AND METHODS: At total of 190 patients were randomly assigned to receive capecitabine 1000 mg/m(2) twice daily on days 1-14 plus oxaliplatin 130 mg/m(2) on day 1 (CapOx), capecitabine 825 mg/m(2) twice daily on days 1-14 plus gemcitabine 1000 mg/m(2) on days 1 and 8 (CapGem) or gemcitabine 1000 mg/m(2) on days 1 and 8 plus oxaliplatin 130 mg/m(2) on day 8 (mGemOx). Treatment cycles were repeated every three weeks. The primary end point was progression-free survival (PFS) rate at 3 months; secondary end points included objective response rate, carbohydrate antigen 19-9 response, clinical benefit response, overall survival and toxicity. RESULTS: The PFS rate after 3 months was 51% in the CapOx arm, 64% in the CapGem arm and 60% in the mGemOx arm. Median PFS was estimated with 4.2 months, 5.7 months and 3.9 months, respectively (P = 0.67). Corresponding median survival times were: 8.1 months (CapOx), 9.0 months (CapGem) and 6.9 months (mGemOx) (P = 0.56). Grade 3/4 hematological toxicities were more frequent in the two Gem-containing arms; grade 3/4 non-hematological toxicity rates did not exceed 15% in any arm. CONCLUSION: CapOx, CapGem and mGemOx have similar clinical efficacy in advanced PC. Each regimen has a distinct but manageable tolerability profile.

Interferon-gamma (IFN-gamma)- and tumour necrosis factor (TNF)-induced nitric oxide as toxic effector molecule in chronic dextran sulphate sodium (DSS)-induced colitis in mice.

Excess nitric oxide formation caused by the activity of the inducible nitric oxide synthase has been implicated as a toxic effector molecule in the pathogenesis of experimental colitis and inflammatory bowel disease. It was therefore investigated whether inhibition of this synthase or the cytokines TNF and IFN-gamma, inducers of nitric oxide synthase, had effects on chronic colitis in mice. Chronic colitis was induced in mice by repeated feeding of DSS. Cytokines were neutralized by treatment with MoAbs and nitric oxide synthase was inhibited by aminoguanidine. The degree of colonic inflammation was assessed by a histological score and colon length. Aminoguanidine treatment reduced nitric oxide activity by 60% (P = 0. 0004), the histological score by 31% (P = 0.005) and increased colon length by 1.4 cm (P = 0.002). Neutralization of TNF and IFN-gamma resulted in increased colon length (0.7 cm, P = 0.07 and 0.8 cm, P = 0.03), improved histological score (19%, P = 0.045 and 25%, P = 0. 013), and reduced nitric oxide activity (31%, P = 0.07 and 54%, P = 0.004) compared with controls. The combination of anti-cytokine treatments had additive effects. TNF and IFN-gamma are involved in perpetuation of chronic DSS-induced colitis, and induction of excessive nitric oxide activity could be their common effector mechanism.

Neutralization of tumour necrosis factor (TNF) but not of IL-1 reduces inflammation in chronic dextran sulphate sodium-induced colitis in mice.

The cytokines TNF and IL-1 have been implicated as mediators of the inflammatory processes in patients with inflammatory bowel disease (IBD). To investigate the role of these cytokines in mucosal inflammation we used anti-cytokine strategies in a mouse model of acute and chronic colitis. Mice which received 5% dextran sulphate sodium (DSS) in their drinking water showed signs of acute colitis on day 4, with severe weight loss and bloody diarrhoea. Chronic colitis was established after four cycles of feeding 5% DSS for 7 days and water for 10 days, with the mice showing diarrhoea but no weight loss. In acute colitis, treatment with anti-IL-1 reagents, anti-TNF MoAb, or dexamethasone (DEX) led to aggravation. By contrast, in chronic colitis, treatment of mice with several IL-1 activity-inhibiting reagents failed to show significant effects, whereas anti-TNF MoAb or DEX significantly reduced the colitis. We conclude that in acute colitis IL-1 and TNF are beneficial, whereas in chronic colitis, TNF but not IL-1 seems to play a major role in perpetuation of chronic inflammation.

Initial clinical results with technetium-99m-labeled LL2 monoclonal antibody fragment in the radioimmunodetection of B-cell lymphomas.

BACKGROUND: Various monoclonal antibodies (MoAb) labeled with Iodine-131 or Indium-111 (In-111) have been investigated for radioimmunodetection of Hodgkin's and non-Hodgkin's lymphomas. Successful radioimmunotherapy also has been reported. The purpose of this pilot study was to stage non-Hodgkin's B-cell lymphomas (NHL) using whole body scintigraphy with technetium-99m (Tc-99m)-labeled murine monoclonal antibody LL2 (EPB-2) Fab' (Immunomedics, Morris Plains, NJ). Others have shown this MoAb to have specific binding to B-cell lymphomas by flow cytometry and immunofluorescence. Initial clinical studies by others have demonstrated targeting of NHL with the Tc-99m-labeled LL2-Fab'. METHODS: One milligram of the antibody was injected intravenously after being radiolabeled with 30 mCi Tc-99m. Fifteen patients with high (n = 6), low (n = 2), and intermediate (n = 7) grade NHL were studied. No adverse effects were noted. Planar whole body imaging and single-photon emission computed tomography were performed at 2-6 h and 20-24 h postinjection. Human anti-mouse antibody levels were determined before injection and at 2 and 6 weeks. RESULTS: In 4 of 15 patients (27%), the disease stage was altered in response to the scintigraphic findings. The physiologic biodistribution of the antibody demonstrated splenic uptake caused by antibody targeting of the white pulp and of normal B-cells, and renal uptake caused by urinary excretion. Lymph node and bone marrow involvement of known tumor sites were clearly seen. A number of previously unknown tumor sites were revealed by LL2-radioimmunodetection despite normal morphologic imaging results. Long-term follow-up of these patients is required to verify these findings. No human anti-mouse antibody elevations or adverse reactions were found in the patients studied. CONCLUSION: These preliminary data suggest that Tc-99m-labeled LL2 Fab' yields useful clinical results, especially for the staging of patients with NHL before initial therapy or for the detection of early disease recurrence.

Distribution and infection of Langerhans cells in the skin of HIV-infected healthy subjects and AIDS patients.

The in situ content of cells of the reticuloendothelial system and lymphatic cells was examined in the skin of eight symptom-free HIV-positive individuals, three AIDS patients and eleven healthy immunocompetent volunteers. The epidermis was obtained in vivo by the suction blister technique. The numbers of CD68+, CD3+, CD8+, CD25-(IL2R)+ and HLA-DR+ intraepidermal cells proved to be independent of the number of CD4+ peripheral blood lymphocytes. At the same time, the intraepidermal concentrations of these cells were generally low in symptom-free HIV-infected individuals. The strong inverse correlation between the number of epidermal Langerhans cells (LC) and the severity of immunodeficiency was quantitatively confirmed; an increase in LC in symptom-free HIV-infected individuals was found. Thus, the reduction in these cells which was observed in the epidermis of AIDS patients began at a significantly elevated level. In contrast to results from other studies, in AIDS patients, in the present study, the concentration of epidermal LC did not differ significantly from that of healthy immunocompetent volunteers. The immunohistochemical technique can be as effective as in situ hybridization for the detection of HIV in the skin. Our results suggest that the viral load of the skin is rather low in HIV-infected subjects. HIV was demonstrated in one cell of one AIDS case by in situ techniques and this result was confirmed by a polymerase chain reaction examination using the same amount of tissue as for the in situ techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of human recombinant interferon-alpha and interferon-gamma on bone marrow progenitor cells of HIV-positive individuals.

As a result of a pathophysiologically unexplainable bone marrow failure, most patients with progressive stages of human immunodeficiency virus (HIV) infection develop anemia, leukopenia, and thrombocytopenia. Besides the possibility of immune-mediated cytolysis or of direct viral infection of hemopoietic progenitor cells, the inhibitory influence of cytokines, for example interferon-alpha (IFN-alpha) and IFN-gamma, on hemopoiesis of HIV-infected patients might be considered as one parameter that contributes to myelosuppression. Therefore, progenitor cells from the bone marrow of HIV+ and HIV- persons were exposed to increasing concentrations of recombinant human IFN-alpha and IFN-gamma in methylcellulose assays. The colony formation of pluripotent (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells was inhibited by both interferons. The 50% inhibitory doses (ID50) of IFN-alpha were 125.6 U/mL and 131.5 U/mL for BFU-E from HIV-infected persons and normal controls, respectively; the corresponding ID50 of IFN-alpha for CFU-GM growth was 1095.8 U/ml and above 3000 U/ml. When IFN-gamma was studied the ID50 was 341.7 and 2794.6 U/ml for BFU-E from HIV-infected and healthy individuals, respectively, while the ID50 for CFU-GM was above the highest dose levels in both groups (greater than 3000 U/ml). The ID50 for CFU-GEMM was below the lowest dose levels of IFN alpha and IFN gamma tested in both groups (less than 10 U/ml). The inhibitory effects could be specifically neutralized by monoclonal antibodies against IFN-alpha and IFN-gamma, thus confirming that the suppressive effects were due to the cytokines used.

TIBO R82913, a new HIV-1 inhibiting agent, does not inhibit hematopoietic progenitor cells.

In progressive stages of infection with human immunodeficiency virus type 1 (HIV-1), the majority of patients develop a pathophysiologically not yet completely explainable bone marrow failure with anemia, leukopenia, and thrombocytopenia. The clinically most widely used HIV-inhibiting antiviral drugs azidothymidine (AZT) and dideoxyinosine (ddI) frequently are hematotoxic to the host, resulting in dose reduction or discontinuation of antiviral therapy. In recent studies, a novel series of benzodiazepine derivatives highly active against HIV-1 was synthesized. These antiviral compounds have a much more favorable therapeutical index than the well-known 2'3'-dideoxyribosides, like AZT. In the experiments presented here, the authors investigated the most promising derivative R82913 [(+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl- 6-(3-methyl-2-butenyl)-imidazo[4,5,1-jk] [1,4]-benzodiazepin-2(1H)-thione] (TIBO) with regard to its toxicity on bone marrow-derived hematopoietic progenitor cells from six HIV-1+ and HIV- persons, respectively. In methylcellulose assays for hematopoietic colony growth any hematotoxic effects of R82913 in vitro were excluded, as both groups showed no difference of progenitor cell growth with or without the TIBO derivative, even at concentrations 6.7 x 10(4) times higher than the 50% inhibitory concentration for cytopathicity by HIV-1.

Effect of recombinant human transforming growth factor beta and tumor necrosis factor alpha on bone marrow progenitor cells of HIV-infected persons.

With progressive disease, the majority of patients with human immunodeficiency virus (HIV) infection develop bone marrow failure with anemia, leukopenia, and thrombocytopenia, the cause of which has not yet been clarified. Besides direct infection of bone marrow progenitor cells and immune-mediated cytolysis, the action of inhibitory cytokines, like transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha), has to be discussed with regard to their pathophysiological role in HIV-induced bone marrow failure. Therefore, the influence of recombinant human TGF-beta and TNF-alpha on colony growth of pluripotent (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells from the bone marrow of HIV-1-infected persons and normal controls was assessed in methylcellulose cultures. Both cytokines inhibited the colony formation of hematopoietic progenitor cells from HIV-positive persons. When added to unseparated bone marrow cells from HIV-infected persons and normal controls, the 50% inhibition (ID50) of BFU-E by TGF-beta occurred at 1.3 ng/ml and 3.7 ng/ml, respectively, while the ID50 of CFU-GM occurred at 15.5 ng/ml and 142.7 ng/ml. Concentrations of TNF-alpha, causing 50% inhibition of colony formation by bone marrow cells from HIV-infected or noninfected individuals were 6.3 U/ml and 17.0 U/ml for BFU-E, and 24.4 U/ml and greater than 3,000 U/ml for CFU-GM, respectively. The ID50 of the CFU-GEMM growth was below the lowest concentration of both cytokines tested. The suppressive effects were specifically abolished by antibodies against TGF-beta and TNF-alpha, thus confirming that the inhibitory activities were due to the cytokine preparation used.

Infection of granulocyte/monocyte progenitor cells with HIV1.

In order to study whether cytopathic HIV1 infection of haemopoietic progenitor cells is involved in the derangement of haemopoiesis in patients with HIV1 infection, we infected enriched progenitor cells with HIV1, by addition of viral inoculate supernatants from HIV1-infected peripheral blood mononuclear cells or by coculture with HIV1-infected monocytes/macrophages. Progenitor cells were seeded into colony assays and single colonies were chosen for HIV1 mRNA determination by in situ hybridization. Growth of progenitors was not affected by infection. However, up to 42% of colonies of pluripotent progenitor cells (colony-forming unit/granulocyte-erythrocyte-monocyte; CFU-GEM) and committed progenitor cells CFU/granulocyte-monocyte (CFM-GM) contained HIV1 mRNA-expressing cells. In addition, we studied HIV1 infection of progenitor cells from the bone marrow of 6 patients with AIDS or AIDS-related complex. Two patients were negative, two had a few colonies expressing HIV1 mRNA in a minority of cells, and in the remaining two, up to 11% of CFU-GM contained HIV1-expressing cells. Thus, infection of progenitor cells with HIV1 was achieved experimentally in vitro and occurs in vivo. However, growth of progenitors after in vitro infection continues and therefore HIV1 infection does not seem to contribute directly to the reduced incidence of haemopoietic progenitor cells in vivo.

Internalization of interleukin 1 (IL 1) correlates with IL 1-induced IL 2 receptor expression and IL 2 secretion of EL4 thymoma cells.

The cytokine interleukin 1 (IL 1) plays an important role in the induction of IL 2 secretion and high-affinity IL 2 receptor (IL 2R) expression by T cells. The events that follow binding of IL 1 to IL 1R, however, are still unknown. In this study we describe two variants of the murine thymoma EL4 (5D3 and D6/76) that express comparable numbers of cell surface IL 1 receptors and bind IL 1 with the same affinity, but show distinct IL 1-dependent IL 2 secretion and IL 2R expression. In the presence of the tumor promoter phorbol 12-myristate 13-acetate IL 1 augments IL 2 secretion and IL 2R expression of EL4 5D3 but not of EL4 D6/76 cells. Comparison of the internalization of IL 1 by both clones revealed that EL4 D6/76 was unable to transport cell surface-bound IL 1 to the cytoplasm. These findings suggest that internalization of receptor-bound IL 1 is required for the action of this cytokine.