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Ulrike Olszewski, Ernst Ulsperger, Klaus Geissler, Gerhard HamiltonLudwig Boltzmann Institute of Clinical Oncology and Photodynamic Therapy, Ludwig Boltzmann Cluster of Translational Oncology, Vienna, AustriaAbstract: Platinum(IV) coordination complexes like oxoplatin (cis,cis,trans-diammine-dichlorido-dihydroxido-platinum[IV]) show high stability and therefore can be utilized orally for outpatient care. Although oxoplatin is capable of binding directly to DNA after prolonged incubation, platinum(IV) agents are considered to be largely inert prodrugs that are converted to highly cytotoxic platinum(II) compounds by reducing substances, enzymes, or microenvironmental conditions. Reaction of oxoplatin with 0.1 M hydrogen chloride mimicking gastric acid yields cis-diammine-tetrachlorido-platinum(IV) (DATCP[IV]), which exhibits two-fold increased activity. The presence of chlorides as ligands in the axial position results in a high reduction potential that favors transformation to platinum(II) complexes. In this study, the intracellular effect of the highly reactive tetrachlorido derivative was investigated in comparison with an equipotent dose of cisplatin. Genome-wide expression profiling of NCI-H526 small cell lung cancer cells treated with these platinum species revealed clear differences in the expression pattern of affected genes and concerned cellular pathways between DATCP(IV) and cisplatin. Application of DATCP(IV) resulted in extensive downregulation of protein and ATP synthesis, cell cycle regulation, and glycolysis, in contrast to cisplatin, which preferentially targeted glutathione conjugation, pyruvate metabolism, citric acid cycle, and the metabolism of amino acids and a range of carbohydrates. Thus, the oxoplatin metabolite DATCP(IV) constitutes a potent cytotoxic derivative that may be produced by gastric acid or acidic areas prevailing in larger solid tumors, depending on the respective pharmaceutical formulation of oxoplatin. Furthermore, DATCP(IV) exhibits intracellular effects that are clearly different from the expected reduced product cisplatin(II). In conclusion, activation of the platinum(IV) complex oxoplatin seems to involve the generation of a cytotoxic six-coordinate species, dependent on prevailing conditions, and its effects need to be considered in addition to the effects of the potential final platinum(II) product.Keywords: platinum, oxoplatin, metabolites, small cell lung cancer, cell line, gene expression, microarray

Aim: Coagulation is frequently activated in cancer patients and has been correlated with an unfavorable prognosis. To evaluate whether a putative release of tissue factor (TF) by circulating tumor cells (CTCs) represents a target to impair the dissemination of small cell lung cancer (SCLC), the expression of relevant proteins in a panel of permanent SCLC and SCLC CTC cell lines that have been established at the Medical University of Vienna., Methods: Five CTC and SCLC lines were analyzed using a TF enzyme-linked immunosorbent assay (ELISA) tests, RNA sequencing, and western blot arrays covering 55 angiogenic mediators. Furthermore, the influence of topotecan and epirubicin as well as hypoxia-like conditions on the expression of these mediators was investigated., Results: The results demonstrate that the SCLC CTC cell lines express no significant amounts of active TF but thrombospondin-1 (TSP-1), urokinase-type plasminogen activator receptor (uPAR), vascular endothelial-derived growth factor (VEGF) and angiopoietin-2 in two cases. The major difference between the SCLC and SCLC CTC cell lines found was the loss of the expression of angiogenin in the blood-derived CTC lines. Topotecan and epirubicin decreased the expression of VEGF, whereas hypoxia-like conditions upregulated VEGF., Conclusions: Active TF capable of triggering coagulation seems not to be expressed in SCLC CTC cell lines in significant levels and, thus, CTC-derived TF seems dispensable for dissemination. Nevertheless, all CTC lines form large spheroids, termed tumorospheres, which may become trapped in clots of the microvasculature and extravasate in this supportive microenvironment. The contribution of clotting to the protection and dissemination of CTCs in SCLC may be different from other solid tumors such as breast cancer., Competing Interests: The authors declare that they have no conflicts of interest., (© The Author(s) 2023.)

Although the RAS-pathway has been implicated as an important driver in the pathogenesis of chronic myelomonocytic leukemia (CMML) a comprehensive study including molecular and functional analyses in patients with progression and transformation has not been performed. A close correlation between RASopathy gene mutations and spontaneous in vitro myeloid colony (CFU-GM) growth in CMML has been described. Molecular and/or functional analyses were performed in three cohorts of 337 CMML patients: in patients without (A, n = 236) and with (B, n = 61) progression/transformation during follow-up, and in patients already transformed at the time of sampling (C, n = 40 + 26 who were before in B). The frequencies of RAS-pathway mutations (variant allele frequency ≥ 20%) in cohorts A, B, and C were 30%, 47%, and 71% ( p < 0.0001), and of high colony growth (≥20/10 5 peripheral blood mononuclear cells) 31%, 44%, and 80% ( p < 0.0001), respectively. Increases in allele burden of RAS-pathway mutations and in numbers of spontaneously formed CFU-GM before and after transformation could be shown in individual patients. Finally, the presence of mutations in RASopathy genes as well as the presence of high colony growth prior to transformation was significantly associated with an increased risk of acute myeloid leukemia (AML) development. Together, RAS-pathway mutations in CMML correlate with an augmented autonomous expansion of neoplastic precursor cells and indicate an increased risk of AML development which may be relevant for targeted treatment strategies.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer which disseminates vigorously and has a dismal prognosis. Metastasis of SCLC is linked to an extremely high number of circulating tumor cells (CTCs), which form chemoresistant spheroids, termed tumorospheres. Intravasation and extravasation during tumor spread requires the activity of a number of proteases to disintegrate the stroma and vascular tissue. Generation of several permanent SCLC CTC lines allowed us to screen for the expression of 35 proteases using Western blot arrays. Cell culture supernatants of two CTC lines, namely BHGc7 and 10, were analyzed for secreted proteases, including matrix metalloproteinases (MMPs), ADAM/TS, cathepsins, kallikreins, and others, and compared to proteases expressed by SCLC cell lines (GLC14, GLC16, NCI-H526 and SCLC26A). In contrast to NCI-H526 and SCLC26A, MMP-9 was highly expressed in the two CTC lines and in GLC16 derived of a relapse. Furthermore, cathepsins (S, V, X/Z/P, A and D) were highly expressed in the CTC lines, whereas ADAM/TS and kallikreins were not detectable. In conclusion, SCLC CTCs express MMP-9 and a range of cathepsins for proteolysis and, aside from tissue degradation, these enzymes are involved in cell signaling, survival, and the chemoresistance of tumor cells.

Small cell lung cancer (SCLC) represents 15% of lung cancers and is characterized by early dissemination, development of chemoresistance and a poor prognosis. A host of diverse drugs failed invariably and its mechanisms of global chemoresistance have not been characterized so far. SCLC represents the prototype of an aggressive and highly metastatic tumor which is ultimately refractory to any treatment. High numbers of circulating tumor cells (CTCs) allowed us to establish 5 CTC cell lines (BHGc7, 10, 16, 26 and UHGc5) from patients with recurrent SCLC. These cell lines exhibit the typical SCLC markers and CTCs of all patients developed spontaneously large multicellular aggregates, termed tumorospheres. Ki67 and carbonic anhydrase 9 (CAIX) staining of tumorosphere sections revealed quiescent and hypoxic cells, respectively. Accordingly, comparison of the chemosensitivity of CTC single cell suspensions with tumorospheres demonstrated increased resistance of the clusters against chemotherapeutics commonly used for treatment of SCLC. Therefore, global chemoresistance of relapsing SCLC seems to rely on formation of large tumorospheres which reveal limited accessibility, lower growth fraction and hypoxic conditions. Since similar tumor spheroids were found in other tumor types, SCLC seems to represent a unique tumor model to study the association of CTCs, metastasis and drug resistance.

Small cell lung cancer (SCLC) is a highly malignant disease with dismal prognosis. Although great progress has been made in investigating genetic aberrations and putative drivers of this tumor entity, the mechanisms of rapid dissemination and acquisition of drug resistance are not clear. The majority of SCLC cases are characterized by inactivation of the tumor suppressors p53 and retinoblastoma (Rb) and, therefore, interchangeable drivers will be difficult to target successfully. Access to pure cultures of SCLC circulating tumor cells (CTCs) and study of their tumor biology has revealed a number of new potential targets. Most important, expression of chitinase-3-like-1/YKL-40 (CHI3L1) which controls expression of vascular epithelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) was newly described in these cells. The process switching CHI3L1-negative SCLC cells to CHI3L1-positive CTCs seems to be associated with cytokines released by inflammatory immune cells. Furthermore, these CTCs were found to promote monocyte-macrophage differentiation, most likely of the M2 tumor-promoting type, recently described to express PD-1 immune checkpoint antigen in SCLC. In conclusion, dissemination of SCLC seems to be linked to conversion of regular tumor cells to highly invasive CHI3L1-positive CTCs, which are protected by immune system suppression. Besides the classical targets VEGF, MMP-9 and PD-1, CHI3L1 constitutes a new possibly drugable molecule to retard down dissemination of SCLC cells, which may be similarly relevant for glioblastoma and other tumor entities.

Background: Results of an observational study on docetaxel-based therapy in non-small cell lung cancer (NSCLC) with focus on symptom control and therapy response, are reported., Patients and Methods: A total of 233 patients with NSCLC treated with docetaxel-containing therapy were analyzed., Results: The pre-existing symptoms of cough, dyspnea and pain markedly improved after three cycles of docetaxel-based therapy. Regression of symptoms was strongly associated with therapy response, but unexpectedly, patients with stable disease had also a substantial benefit. Alltogether, the response after three cycles was complete in 0.9% and partial in 26.6% of patients, respectively., Conclusion: Symptom control was achieved in the majority of cases, which received three cycles of docetaxel-based therapy. Thus, a clinical benefit was regularly reached shortly after initiation of chemotherapy.

Background: In epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC), the tyrosine-kinase inhibitor gefitinib is in broad use. We retrospectively analysed data for 82 patients with advanced NSCLC treated with gefitinib and correlated benefits with clinical baseline and therapy-related parameters., Patients: Of all patients 48/82 were male; the median age at start of gefitinib was 67.2 years; 14/58 informative patients were never-smokers; 57/82 patients suffered from adenocarcinoma, including 7 with bronchoalveolar-carcinomas., Results: As to be expected, partial remission was observed in 10% of patients, stable disease in 29%, progression-free survival was 3.1 months and overall survival 9.2 months. Gefitinib was more efficacious in women, never-smokers and patients with bronchoalveolar-carcinoma. Furthermore, anemia and elevated C-reactive protein levels were unfavourable for therapeutic efficacy. Patients developing skin reactions under gefitinib achieved response far more frequently, with longer progression-free survival and overall survival., Conclusion: Basic clinical parameters are good predictors for response to EGFR tyrosine-kinase inhibitor therapy, which may be of value if EGFR mutation status is not available.

Purpose: Dichloroacetate (DCA) inhibits pyruvate dehydrogenase kinase (PDK), and thus promotes glucose oxidation over glycolysis and induces apoptotic death of tumor cells. The present study investigated the potential of DCA to increase the antitumor effects of platinum- based compounds against a panel of permanent cell lines, including small cell lung cancer (SCLC), ovarian cancer, and Ewing's sarcoma in vitro., Methods: DCA at a concentration of 10 mM was combined with cisplatin, carboplatin, satraplatin, the satraplatin metabolite JM118, oxaliplatin, oxoplatin, and picoplatin, and the cytotoxic activity was evaluated in proliferation tests employing a panel of different cell lines. Additionally, cells were pretreated with DCA and then exposed to the platinum drugs and etoposide, or incubated with cisplatin or etoposide followed by application of DCA, respectively., Results: DCA 10 mM significantly increased the cytotoxicity of the platinum-based drugs carboplatin, satraplatin, JM118, and oxoplatin, but not cisplatin, picoplatin, and oxaliplatin in vitro. Preincubation of cell lines with DCA 10 mM for three days reduced the antiproliferative activity of platinum-based agents in sequential application, but exposure of cells pretreated with cisplatin or etoposide to DCA resulted in minor sensitization. The inhibitory effect of DCA showed no correlation with sensitization to the platinum compounds., Conclusion: DCA alone in a concentration that shows low antiproliferative activity is capable of increasing the cytotoxicity of selected platinum compounds upon coincubation, and such combinations may be interesting for clinical application in tumors like SCLC, Ewing's sarcoma, and ovarian cancer refractory to cisplatin chemotherapy as standard care. The mechanism of this synergistic effect of DCA in combination with specific platinum species remains to be investigated.

Platinum(IV) compounds like oxoplatin (cis, cis, trans-diammine-dichlorido-dihydroxido-platinum(IV)) show increased stability and therefore can be applied orally. In a panel of 38 human cancer cell lines this drug induced S-phase arrest and cell death with IC(50) values 2.5-fold higher than cisplatin. Oxoplatin may be converted to cisplatin by intracellular reducing agents, however, exposure to 0.1 M HCl mimicking gastric acid yielded cis-diammine-tetrachlorido-platinum(IV) exhibiting twofold increased activity. Similar results were obtained for another platinum(IV) compound, JM 149 (ammine-dichlorido-(cyclohexylamine)-dihydroxido-platinum(IV)), but not for its parent drug JM 216/satraplatin. Genome-wide expression profiling of H526 small cell lung cancer cells treated with these platinum species revealed clear differences in the expression pattern of affected genes between oxoplatin and cisplatin. In conclusion, oxoplatin constitutes a potent oral agent that is either reduced or converted to distinct active compounds, for example, by gastric acid or acidic areas prevailing in solid tumors, in dependence of the respective pharmaceutical formulation.

It would be of benefit for the clinical relevance of tumor marker determination to be demonstrated, as a lot of markers are now in clinical use. Increased levels of carcinoembryonic antigen correlate with the stage of breast carcinoma. CA 15-3 should also be measured during follow-up of patients with this disease. The latest findings suggest a higher sensitivity and specificity of CA 15-3 than of CEA. The prognostic value and the usefulness of CEA measurement in screening seem to be poor. The measurement of CA 125 seems to be a reliable method for monitoring the presence and clinical behavior of ovarian cancer. It is suggested that invasive diagnostic procedures are not required in patients with normal marker levels. The management of chorion carcinoma can be determined as an ideal model in the range of marker application. Only in this disease does the marker HCG reach almost 100% sensitivity and specificity. The definition of response to chemotherapy and the appearance of relapse can be based on HCG measurement.