Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T-cell-induced immune escape mechanism.

Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.

A phase Ib study of pertuzumab, a recombinant humanised antibody to HER2, and docetaxel in patients with advanced solid tumours.

Pertuzumab represents the first in a new class of targeted therapeutics known as HER dimerisation inhibitors. We conducted a phase Ib study to determine the maximum-tolerated dose, the dose limiting toxicities (DLT), and pharmacokinetic (PK) interaction of docetaxel when administered in combination with pertuzumab. Initially, two dose levels of docetaxel (60 and 75 mg m(-2)) were explored in combination with a fixed dose of 1050 mg of pertuzumab; then two dose levels of docetaxel (75 and 100 mg m(-2)) were explored in combination following a fixed dose of 420 mg of pertuzumab with a loading dose of 840 mg. Both drugs were administered intravenously every 3 weeks. The latter dose of pertuzumab was allowed after an amendment to the original protocol when phase II data suggesting no difference in toxicity or activity between the 2 doses became available. Two patients out of two treated at docetaxel 75 mg m(-2) in combination with pertuzumab 1050 mg suffered DLT (grade 3 diarrhoea and grade 4 febrile neutropaenia). Two out of five patients treated at docetaxel 100 mg m(-2) in combination with pertuzumab 420 mg with a loading dose of 840 mg suffered DLT (grade 3 fatigue and grade 4 febrile neutropaenia). Stable disease was observed at four cycles in more than half of the patients treated and a confirmed radiological partial response with a >50% decline in PSA in a patient with hormone refractory prostate cancer were observed. There were no pharmacokinetic drug-drug interactions. The recommended phase II dose of this combination was docetaxel 75 mg m(-2) and 420 mg pertuzumab following a loading dose of 840 mg.

Chronic recurring infarction of the spleen: sonographic patterns and complications.

PURPOSE: Splenic infarction is a major problem of splenic pathology but is characterized by a high tendency for complete healing. The purpose of this study is to describe frequency, sonographic patterns, and complications of chronic infarction (CI) METHODS: Between 1980-2001 550 patients with focal splenic lesions were diagnosed by ultrasound. Eighty patients had an acute infarction, and in 14 cases a chronic infarction was diagnosed and confirmed by cytohistology/splenectomy (n = 3) or sonographic follow-up examination (n = 11). All patients with Cl had been investigated by B-mode sonography and colour Doppler sonography (CDS). Data were retrospectively evaluated. RESULTS: Two types of Cl could be discriminated. Type I morphology (n =8) was predominantly found in homozygous sickle-cell anaemia (n= 6) and sonographically characterized by a small or normal sized spleen (n = 6), with diffuse enhanced echogenicity (n= 8), and foci with diminished echogenicity (n=5). Type II morphology (n = 6) was predominantly found in myeloproliferative diseases (n = 4) and characterized by an enlarged spleen with a homogeneous echotexture (n = 7), and a solitary (n = 6), triangular (n=4), hyperechoic (n=4) splenic foci near the splenic surface. On CDS CI were characterized by absent flow signals (n = 7) or by reduced flow signals (n= 7). Spontaneous splenic ruptures occurred as infarction related complications in 3 of 14 cases (21%). CONCLUSIONS: CI develops in 17.5% of patients with infarctions. It occurs predominantly in patients with sickle-cell anaemia and myeloproliferative disease. Two characteristic morphologic patterns were found and associated with an increased risk of spontaneous splenic rupture: Therefore sonographic follow-ups might be useful.

Spontaneous rupture of the spleen: ultrasound patterns, diagnosis and follow-up.

Spontaneous rupture of the spleen is an extremely rare complication usually of infectious diseases or disorders of the haematopoietic system and has been described mostly in case reports. The incidence, symptoms, causes, therapy, and prognosis are poorly defined. From July 1985 to January 2000 41 patients with spontaneous splenic rupture were diagnosed by abdominal ultrasound and confirmed by splenectomy (n=12), CT (n=15), and ultrasound follow up (n=26). An ultrasound grading system was retrospectively established based on the degree of splenic injury (grade 0-2=low grade injury, grade 3=high grade injury) and correlated with surgical procedures. 30 day mortality rate was studied in relation to underlying disorders, ultrasound grades and treatment decisions. 21 patients had underlying malignant disorders (group I) and 20 patients had benign diseases (group II). Between group I and II we observed a highly significant difference in 30 day mortality rates (n=7; 38.1% vs n=1; 5%, p<0.01), but no significant difference in high grade injury rate (n=3; 14.3% vs n=2; 10.0%; p=ns) and surgical treatment rate (n=5; 23.8% vs n=7; 35.0%; p=ns). Depending on ultrasound grades the surgical procedures were 0% for grade 0, 16.7% for grade 1, 30.4% for grade 2, and 60% for grade 3. There were no significant differences between patients, who died within the first 30 days (n=9) and those who survived more than 30 days (n=32) regarding high grade splenic injury rate (n=0; 0% vs n=5; 15.6%; p=ns), and surgical treatment rate (n=2; 22.2% vs n=10; 31.2%; p=ns). Spontaneous rupture of the spleen is an extremely rare event. It is associated with a high mortality rate within 30 days in patients with malignant disease. Sonomorphologic grading is helpful for treatment decisions. 30 day mortality rate is correlated with neither ultrasound grades, nor surgical treatment rates.

Marked increase of the growth factors pleiotrophin and fibroblast growth factor-2 in serum of testicular cancer patients.

BACKGROUND: Malignant tumors of the testis are among the most common cancers in men between the ages of 15 and 30 years. The sensitivity of detection of known tumor markers depends upon the tumor histology and stage. In other cancers, increased serum concentrations of various angiogenic growth factors have been described as potential markers for tumor progression and metastasis. One main histological feature of testicular cancer is profound angiogenesis. DESIGN: In this study, we investigated by sensitive enzyme-linked immunosorbent assays (ELISAs) the levels of various growth and angiogenesis factors in the serum of testicular cancer patients as compared with normal control subjects. For the most profoundly increased growth factors, pleiotrophin (PTN) and fibroblast growth factor-2 (FGF-2), we furthermore analyzed tumor lysates by northern blotting, RT-PCR and ELISA. RESULTS: We demonstrate a marked elevation of average serum levels of PTN ( approximately 20-fold) and of FGF-2 ( approximately 7-fold) in patients and expression of both growth factors in tumor biopsies. To a lesser extent, vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) serum levels were increased, whereas FGF-4 and transforming growth factor-beta levels were similar to those in normal control subjects. Elevation of PTN, FGF-2, EGF and VEGF was detected in seminomatous as well as non-seminatous tumors, and even in early stages. CONCLUSIONS: PTN and FGF-2 may represent promising new diagnostic markers for testicular cancer with high sensitivity even in early-stage testicular cancer. Further studies are warranted to extend our analyses.

[AFP-producing adenocarcinoma of the stomach. A rare tumor with poor prognosis]. / Das AFP-positive Adenokarzinom des Magens. Ein seltener Tumor mit früher Disseminierung.

A 27-year-old, previously healthy man with abdominal discomfort was diagnosed with a small gastric tumor of the cardia by means of gastroscopy. Further staging revealed diffuse hepatic metastases and enlarged mediastinal lymph nodes. Serum alpha-fetoprotein (AFP) was grossly increased (7179 micro g/l). Biopsies taken from the gastric tumor and one of the hepatic metastases revealed a poorly differentiated adenocarcinoma (grade 3) with papillary and small solid areas and frequent clear cells. Cytoplasmic hyaline droplets were positive with PAS staining (diastase-resistant). Immunohistochemistry revealed focal tumor cells strongly positive for AFP and there was luminal expression of CEA. The diagnosis of an AFP-producing adenocarcinoma of the stomach was made. In spite of intensive combination chemotherapy the patient succumbed to his disease 3 months after diagnosis. The rare AFP-producing adenocarcinoma of the stomach is characterised by a distinct morphology and immunohistochemistry. A hepatoid differentiation may occur but is not obligatory as this case shows. In differential diagnosis, a metastasising germ cell tumor should be excluded. The prognosis for an AFP-positive adenocarcinoma is poor.

Serum levels of the angiogenic factor pleiotrophin in relation to disease stage in lung cancer patients.

Pleiotrophin is a heparin-binding growth factor involved in the differentiation and proliferation of neuronal tissue during embryogenesis, and also secreted by melanoma and breast carcinoma cells. Pleiotrophin exhibits mitogenic and angiogenic properties and has been shown to influence the vascular supply, expansion and metastasis of tumour cells. Our aim was to study the serum and plasma concentrations of pleiotrophin and the classical angiogenic growth factor vascular endothelial growth factor. Using a specific ELISA-test we studied patients with small cell lung cancer (n=63), and patients with non-small cell lung cancer (n=22) in comparison to healthy control subjects (n=41). In most of the lung cancer patients (81%), we found serum levels of pleiotrophin above those of control subjects (P<0.001). Of the 63 small cell lung cancer patients in the study pleiotrophin serum levels were elevated in 55 cases (87%) and in 14 cases (63%) of the 22 non-small cell lung cancer patients. Pleiotrophin mean serum concentrations were 10.8-fold higher in the tumour patient group as compared to the control group (P<0.001). Furthermore, pleiotrophin serum levels correlated positively with the stage of disease and inversely with the response to therapy. Plasma vascular endothelial growth factor concentrations were elevated in only in 28.6% of small cell lung cancer and 45.5% of non-small cell lung cancer patients by an average of 2.3-fold. Quite strikingly, there was no apparent correlation between the plasma vascular endothelial growth factor concentration and the stage of disease. Our study suggests that pleiotrophin may be an early indicator of lung cancer and might be of use in monitoring the efficacy of therapy, which needs to be confirmed by larger studies.

Polysulfated heparinoids selectively inactivate heparin-binding angiogenesis factors.

Angiogenesis is a prerequisite for tumor expansion and metastasis. The angiogenic potential of the heparin-binding growth factors acidic fibroblast growth factor (FGF) and basic FGF has been demonstrated in various publications. We studied the inhibitory effects of suramin and the polysulfated heparinoids pentosan polysulfate, dextran sulfate, and fucoidan on the action of FGF. As an experimental model, we used the adrenal cancer cell line SW 13, whose anchorage-independent growth depends on the presence of FGF. The polysulfated heparinoids inhibited FGF-induced growth and binding to the receptor at an IC50 of 0.5-3 micrograms/ml. Suramin inhibited FGF at an IC50 of 100 micrograms/ml. The polysulfated heparinoids exerted no effect on IGF-1 or TGF alpha-related growth. Suramin inhibited the anchorage-independent growth induced by IGF-1 or TGF alpha only at an IC50 of 100 micrograms/ml. Our results indicate that suramin inhibits growth factors in a nonselective way. By contrast, polysulfated heparinoids exert a selective inhibitory effect on heparin binding angiogenesis factors at an IC50, which is 100 times below the IC50 of suramin. Therefore, the administration of polysulfated heparinoids might become a novel approach to tumor therapy based on blocking angiogenesis.

Differential expression and biological activity of the heparin-binding growth-associated molecule (HB-GAM) in lung cancer cell lines.

The growth of human lung cancer cells is regulated positively and negatively by a variety of growth factors through autocrine as well as paracrine mechanisms. In the present report, we studied the differential role and expression of a neuropolypeptide growth factor in 26 lung cancer cell lines. Expression of the heparin-binding growth-associated molecule (HB-GAM) in 12 small cell lung cancer (SCLC) cell lines was compared to that in 14 non-small cell lung cancer (NSCLC) cell lines. HB-GAM mRNA was expressed in 9 of 12 SCLC and 3 of 14 NSCLC cell lines as determined by RT-PCR analyses. Normal human bronchial epithelial cells were used as negative controls. All cell lines which expressed HB-GAM mRNA produced HB-GAM protein as well. Western blot analysis showed that the tumor cells secreted HB-GAM into the media. HB-GAM, purified from lung cancer cell lines, exerted biological activity on fibroblasts, endothelial cells and SW13 cells as determined by thymidine incorporation and soft agar cloning assays. In addition, the biological activity of HB-GAM was blocked by a specific antibody in a dose-dependent way. Our findings suggest that HB-GAM may serve as a marker for SCLC cell lines and that it may function as a paracrine growth factor in human lung cancer. HB-GAM may be a further member of the network of growth factors involved in proliferation, angiogenesis and metastasis of lung tumors.

Growth-inhibitory effects of vitamin D analogues and retinoids on human pancreatic cancer cells.

Retinoids and vitamin D are important factors that regulate cellular growth and differentiation. An additive growth-inhibitory effect of retinoids and vitamin D analogues has been demonstrated for human myeloma, leukaemic and breast cancer cells. We set out to study the effects of the vitamin D analogue EB1089 and the retinoids all-trans- and 9-cis-retinoic acid on the human pancreatic adenocarcinoma cell lines Capan 1 and Capan 2 and the undifferentiated pancreatic carcinoma cell line Hs766T. The cell lines investigated expressed vitamin D receptor, retinoic acid receptor (RAR)-alpha and gamma as determined by polymerase chain reaction after reverse transcription. RAR-beta was expressed only in Hs766T cells. Addition of all-trans-retinoic acid increased the amount of RAR-alpha mRNA in the three cell lines and induced RAR-beta mRNA in Capan 1 and Capan 2 cells. All-trans-retinoic acid at a concentration of 10 nM inhibited the growth of Capan 1 and Capan 2 cells by 40% relative to controls. 9-cis-Retinoic acid was less effective. Neither all-trans-retinoic acid nor 9-cis-retinoic acid affected the growth of Hs766T cells. EB1089, if added alone to the cells, did not significantly inhibit growth. However, the combination of 1 nM EB1089 with 10 nM all-trans-retinoic acid exerted a growth-inhibitory effect of 90% in Capan 1 cells and of 70% in Capan 2 cells. Our data suggest that vitamin D analogues together with retinoids inhibit the growth of human pancreatic cancer cells. However, in vivo studies are necessary to examine the potential use of retinoids and vitamin D analogues on pancreatic cancer.

Regulation and role of TGF beta production in breast cancer.

The influence of antiestrogens on the secretion of transforming growth factor beta (TGF beta) proteins that have an autoinhibitory potential for human cancer cells was studied in the estrogen-responsive human breast cancer cell line, MCF-7: Antiestrogens induce the secretion of TGF beta-1 via a nontranscriptional pathway; TGF beta-1 itself induces TGF beta-2 by a direct transcriptional mechanism; and TGF beta-2 is a marker of antiestrogen action. This hypothesis was confirmed in a clinical study with 18 patients with advanced metastatic breast cancer. TGF beta-2 plasma levels were measured before and after 4 weeks of treatment with tamoxifen. In the majority of patients who responded to the treatment, increasing TGF beta-2 concentrations were seen under therapy. Patients who did not respond did not show changes in the TGF beta-2 plasma level after 4 weeks of treatment. These results suggest that the sequential analysis of TGF beta-1 in plasma before and under treatment with tamoxifen allows the early identification of patients with antiestrogen resistance.

Hormonal regulation of transforming growth factor beta-2 expression in human prostate cancer.

We have previously shown that a transforming factor-beta species (TGF beta) is a hormonally regulated negative growth factor in estrogen responsive MCF-7 human breast cancer cells. We now demonstrate that androgen withdrawal leads to a significant stimulation of TGF beta-2 mRNA in the androgen-responsive human prostate carcinoma cell line LNCaP. These data indicate that TGF beta-2 is a marker of (anti)androgen action in human prostate cancer in vitro. Based on these results we addressed the question of whether TGF beta-2 represented a marker of (anti)androgen action in prostate cancer in vivo: expression of TGF beta mRNA was determined by RNAase protection analysis in normal and malignant prostate tissue obtained from 9 prostate carcinoma patients without endocrine therapy. In parallel, the nuclear dihydrotestosterone (DHT) concentration was measured as an indicator of androgen stimulation in the same tissues. The following results were obtained. Both normal and cancerous tissues show nuclear accumulation of DHT indicating a functional androgen receptor system. TGF beta-2 is equally expressed in both normal and cancerous tissue. Expression of TGF beta-2 and nuclear DHT concentrations are correlated in both benign and malignant tissue. We conclude that TGF beta-2 is a marker of (anti)hormonal action in androgen-dependent tissue.

Detectable inhibition of heparin-binding growth factor activity in sera from patients treated with pentosan polysulfate.

BACKGROUND: Previous studies indicate that the heparinoid pentosan polysulfate (PPS) can inhibit heparin-binding growth factors (HBGFs) released from tumor cells and thus block tumor growth in animal models. However, because of its heparin-like activity, the major toxic effect expected for PPS is its inhibition of coagulation. PURPOSE: Our purpose was to determine if anti-HBGF activity could be achieved in patients without causing complications from anticoagulation. METHODS: We initiated a phase I trial in cancer patients and developed a cell proliferation assay to detect PPS in human serum based on its antigrowth factor activity. Blood samples from six healthy volunteers were collected in tubes containing different concentrations of PPS (FIBREZYM; concentration range, 0-10 micrograms/mL). Additional samples were obtained from four patients in the phase I trial before and after subcutaneous treatment with 15 mg/m2 of PPS. The activated partial thromboplastin time (aPTT), which is associated with coagulation, was measured in all blood samples. Serum prepared from the blood samples was heat inactivated and then incubated for 4-5 days with proliferating SW-13 cells, allowing determination of antigrowth factor activity. RESULTS: PPS added to blood samples increased aPTT only at concentrations above 1 microgram/mL, whereas HBGF-dependent proliferation was inhibited at less than 0.1 microgram/mL. Sera obtained from patients up to 4 hours after PPS treatment specifically inhibited HBGF-dependent cell proliferation by more than 65% even at a 1:10 dilution. At the same time, the aPTT was not altered in these patients, indicating no significant effect on coagulation by this dose of the heparinoid. CONCLUSIONS: HBGF-inhibitory concentrations of PPS can be achieved in patients' sera without significant effects on coagulation. IMPLICATION: The assay presented here could be useful to determine doses and scheduling of treatment in studies evaluating PPS as an antitumor agent.

Increased risk of acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) in a county of Hesse, Germany.

The incidence of acute and chronic myelogenous leukemia has been compared for the two neighbouring regions of Marburg and Giessen in Hesse (Germany). The investigation was based on the incident cases of the years 1983-1989 which have been diagnosed in the hematological departments of the universities of the two regions. The epidemiological evaluation of the data has been carried out in terms of a historical follow-up study, and shows an increased relative risk for the region around Marburg with a particular elevation for one community within this region. Potential determinants are discussed and focus on trinitrotoluene (TNT) and decomposition products which are known to contaminate the soil of this community, in some places severely, due to insufficient removal of remnants of the TNT production in large underground plants during World War II.

Inhibition by pentosan polysulfate (PPS) of heparin-binding growth factors released from tumor cells and blockage by PPS of tumor growth in animals.

BACKGROUND: In a proliferating tumor, locally secreted polypeptide growth factors, which have autocrine and paracrine functions, induce vascularization essential for tumor growth and metastasis. These growth factors may serve as targets for tumor therapy. We have shown that the heparinoid pentosan polysulfate (PPS) can block growth of subcutaneous human tumor xenografts in nude mice and angiogenesis induced by the heparin-binding, Kaposi's sarcoma-derived fibroblast growth factor (K-FGF). PURPOSE: The purpose of this study was to determine whether PPS might also interfere with stimulation of endothelial cells by other growth factors released from tumor cells and whether the promising antitumor effects of PPS extend to other human tumor cell lines. We studied the effects of PPS on stimulation by heparin-binding growth factors released from seven human tumor cell lines in vitro and on tumors growing from these cell lines in athymic nude mice. METHODS: Seven human cell lines established from breast, prostate, epidermoid, and lung carcinomas and rhabdomyosarcoma were used in in vivo as well as in vitro studies of the effects of PPS. We also studied in vitro the effects of PPS on growth factor-induced colony formation of normal rat kidney fibroblasts and human adrenal carcinoma cells. RESULTS: The tumor cell lines released growth factors into their media that stimulated growth of endothelial and epithelial cells as well as fibroblasts. Heparin-affinity chromatography showed that heparin-binding growth factors contributed substantially to this paracrine activity and that PPS inhibited this stimulus. Six of the seven tumor cell lines were resistant to PPS in soft-agar cloning assays and hence did not appear to depend on autocrine stimulation by the heparin-binding growth factors. In contrast to this in vitro resistance, subcutaneous growth of tumors from all cell lines in athymic nude mice was inhibited in a dose-dependent fashion by daily intraperitoneal injections of PPS. CONCLUSIONS: We conclude that heparin-binding growth factors contribute substantially to tumor growth in vivo and that PPS acts by blocking the paracrine effects of heparin-binding growth factors released from the tumor cells. IMPLICATION: PPS could become a novel treatment tool targeting tumor growth factors.