Jutta A. Kurbacher

Use of an ex vivo ATP luminescence assay to direct chemotherapy for recurrent ovarian cancer.

Chemotherapy for recurrent ovarian carcinoma (ROC) produces response rates of 10-80% depending on the prevalence of platinum resistance. Most patients relapse within 1 year and median progression-free survival (PFS) is generally no more than 6 months. Previous pretherapeutic chemosensitlvity assays mostly failed to improve the outcome of patients with ROC. Newly developed ATP assays show promising retrospective correlation with clinical outcome. We report here the first results of ATP assay-directed chemotherapy in patients with ROC. Therapy was selected by the ATP tumor chemosensitivity assay (ATP-TCA) in a prospective open-label pilot trial for ROC. Objective response rate (ORR), PFS and overall survival (OAS) of the first 25 evaluable patients were retrospectively compared with those of 30 others having similar characteristics who were treated empirically within the same period. The actuarial median observation times were 80 weeks for the ATP-TCA group and 83.5 weeks for the control group, respectively. In the control group, a 37% ORR [two complete responses (CR) and nine partial responses (PR)] was followed by a median PFS of 20 weeks and a median OAS of 69 weeks, mainly related to the use of single-agent chemotherapy. The ORR in the ATP-TCA group was 64% (eight CR and eight PR) (p=0.04) with the majority of responses (11 of 16) achieved with novel combinations. The median PFS in this group was 50 weeks (p=0.003) and the median OAS was 97 weeks (p=0.145). Survival of responding patients was similar in both groups. Chemotherapy guided by the ATP-TCA produced a greater benefit with regard to both ORR and PFS in platinum-refractory patients. ATP-TCA-directed chemotherapy for ROC compares favorably with chemotherapy chosen by a clinician and often leads to the choice of novel drug combinations. These promising results now warrant confirmation by prospective randomized trials.

In vitro activity of titanocenedichloride versus cisplatin in four ovarian carcinoma cell lines evaluated by a microtiter plate ATP bioluminescence assay

Titanocenedichloride (MKT 4) is a novel anticancer drug with a broad spectrum of activity in mammalian tumors. We investigated the anticancer efficacy of MKT 4 versus cisplatin and its chemomodulation by buthionine sulfoximine (BSO) in four different human ovarian carcinoma (OvCA) cell lines derived from both primary (A2780. OTN 14) and recurrent tumors (SKOV-3 and OV-MZ-1b) using an in vitro microplate ATP bioluminescence assay (ATP-TCA). Sensitivity against cisplatin was higher in A2780 and OTN 14 compared with MKT 4, whereas the opposite was found in SKOV-3 and OV-MZ-1b cells. In A2780, SKOV-3 and OV-MZ-1b, the cytotoxicity of both agents could be effectively improved by BSO with supraadditive effects observed for MKT 4 in all three cell lines. In OTN 14, however, BSO treatment failed to increase the cytotoxicity of both cisplatin and MKT 4. These results suggest antineoplastic activity of MKT 4 in cisplatin-sensitive and mainly in cisplatin-resistant OvCA cells which can be significantly modulated by BSO-mediated glutathione depletion. Since antineoplastic activity of both cisplatin and MKT-4 observed in OTN 14 could not be reversed by BSO, other mechanisms of drug resistance different from the glutathione redox cycle are likely to be important for both metal compounds.

Influence of Human Luteinizing Hormone on Cell Growth and CA125 Secretion of Primary Epithelial Ovarian Carcinomas in vitro

In the present study, the influence of human luteinizing hormone(hLH) on the growth and the CA 125 secretion of primary ovarian carcinoma cell cultures derived from 11 previously untreated patients was investigated. Two different patterns of in vitro growth of unstimulated ovarian carcinoma cells could be detected: 5 cell cultures in which no changes of cell number were observed during an incubation period of 6 days (group A) and 6 cell cultures which grew without any hormonal support (group B). All tumors of group A showed enhanced cell proliferation when hLH was added to the culture medium, reaching a maximum at dosages of 100 mIU/ml on day 2 after incubation, while such an effect was not observed in group B. In most cases, no positive correlation was found between hLH-induced cell growth and CA 125 release. Our findings demonstrated a dose-dependent hLH-induced stimulation of epithelial ovarian tumors in vitro.

Heterogeneity ofin vitro chemosensitivity in perioperative breast cancer cells to mitoxantroneversus doxorubicin evaluated by a microplate ATP bioluminescence assay

SummaryApart from clinical trials, mitoxantrone (MX) is rarely used in breast cancer (BC) due to the anticipated anthracycline cross-resistance. We have examined this drug versus doxorubicin (DOX) using data obtained fromin vitro microplate ATP tumor chemosensitivity assays (ATP-TCA) of BC cells which were derived from 55 chemotherapy-naive patients at time of primary surgery. Both drugs were tested at 6 different concentrations ranging from 6.25% to 200% peak plasma concentrationin vivo (PPC). Differences between DOX and MX observed for mean IC50, IC90, and a sensitivity index (SI) were not statistically significant.In vitro response rates were 44% for DOX and 52% for MX. 34 of 52 eligible assays (65%) showed comparable activity of both drugs whereas a lack of cross-resistance was observed in the remaining 18 (35%) tumors as indicated by differences for SI. Cumulative concentration-response plots of tumors respondingin vitro with a ≥ 50 percent or ≥ 90 percent tumor cell inhibition showed a strong dose-dependence for both DOX and MX at concentrations which normally can be achieved within clinical tumors (i.e. 6.25%-50% PPC). At higher concentrations, however, cytotoxicity of DOX and MX could not be improved by furtherin vitro dose escalation. Moreover, a substantial proportion of BC specimens (DOX: 48.1%; MX: 40.4%) did not experience a ≥ 90 tumor cell inhibition at 200% PPC. In conclusion,in vitro results obtained by ATP-TCA indicate that there is no cross-resistance between MX and DOX in a substantial proportion of BC patients. This may be clinically useful and suggests that combinations including MX should be tested in patients clinically resistant to DOX containing regimens. Since both drugs produced sigmoidal concentration-response curves, dose escalation beyond a certain point may not produce increased sensitivity.

Isolation and Culture of Ovarian Cancer Cells and Cell Lines

Ovarian carcinomas show considerable heterogeneity of origin, both in terms of site and tissue. The most important and also most frequent of these tumors arise from the coelomic epithelium and are therefore characterized as epithelial ovarian carcinomas (EOC). EOC is often large and advanced at the time of presentation, so that cells are readily obtainable from surgical specimens or effusions. While the primary tumor may be chemosensitive, they often develop resistance and may do so rapidly. Due to the easy access to tumor cells and its biological behavior, EOC is considered to be an ideal model to investigate principal mechanisms of both antineoplastic drug sensitivity and resistance. Although studies on primary EOC cells are now preferred for many of these investigations, EOC cell line studies remain important too. This chapter gives an overview over major techniques required to establish and maintain primary EOC cell cultures and to initiate and cultivate permanently growing EOC cell lines.

Chemosensitivity of normal human trophoblasts evaluated by a newly developed ATP-based luminescence assay.

Trophoblast injury may be one of the possible causes of fetal distress associated with chemotherapy administered during pregnancy. The purpose of this study was to investigate the ex vivo chemosensitivity of normal trophoblasts (NTB) against commonly used antineoplastic agents. Using the newly developed ex vivo ATP-based trophoblast assay (ATP-TBA), 31 NTB freshly sampled from human placentas (gestational week 7–42) were tested against dactinomycin (Act-D), 5-fluorouracil (5-FU), 4-OOH-cyclophosphamide (4-HC), vincristine (VCR) and methotrexate (MTX) alone or in combination with calcium folate (LV). All agents were studied at concentrations relevant to clinical dosages normally used for chemotherapy of solid neoplasms. Of 31 samples studied with the ATP-TBA, 20 (65%) were evaluable. VCR, Act-D and 4-HC were the most active drugs with 55, 45 and 45% of samples responding ex vivo. Antimetabolites were less active, producing ex vivo response rates of 25 (MTX) and 20% (5-FU), respectively. MTX activity was largely neutralized by adding LV. The chemosensitivity of NTB showed considerable inter-individual variations and did not decrease with increasing gestational age. We therefore conclude that NTB of any gestational age exhibit considerable ex vivo sensitivity against common anticancer agents which is comparable to that observed for various solid tumors. The ATP-TBA may be helpful in planning future trials with both single agents and drug combinations in order to standardize and optimize chemotherapy during pregnancy.

Vitamin D-Mangel in der Schwangerschaft: Erste Ergebnisse eines individualisierten Supplementations-Konzepts

Zielsetzung: Mehr als 90% aller Schwangeren weisen im klinischen Alltag ganzjahrig einen initialen Vitamin D (VitD)-Mangel auf (Kettelhoit et al. 2016). Basierend auf diesen Daten wurde ein Serumspiegel-adaptiertes individualisiertes Supplementations-Konzept entwickelt, dessen vorlaufige Ergebnisse hier erstmals prasentiert werden. Methoden: Bei 150 Patientinnen (Pat) wurde bei Erstuntersuchung in der Schwangerschaft der 25-OH-Cholecalciferol (25-OH-CC) Spiegel bestimmt. Darauf basierend erfolgte eine individualisierte VitD-Supplementation. Pat mit 20 ng/ml erfolgte eine VitD-fokussierte Ernahrungsberatung. Eine Kontrolle des 25-OH-CC Spiegels erfolgte 8 – 12 Wochen nach Therapiebeginn nach einer dreitagigen Einnahmekarenz. Ergebnisse: Der mittlere basale 25-OH-CC Spiegel lag bei 9,7 ng/ml, der mittlere Spiegel nach Supplementation betrug 21,8 ng/ml (p 20 ng/ml. Bei den ubrigen 29 Pat wiesen bei Kontrolle 19 (65,5%) 25-OH-CC Werte > 20 ng/ml auf (p = 0,493). Eine VitD-Uberdosierung (> 80 ng/ml) wurde nicht beobachtet. Zusammenfassung: Die individualisierte VitD-Supplementation in der SS ist sicher und effektiv. Die Mehrzahl der Pat wiesen nach 8 – 12 Wochen normalisierte 25-OH-Serumspiegel auf, wobei Uberdosierungen sicher vermieden werden konnten. Der erhebliche Anteil an Pat, die selbst unter intensivierten VitD-Gaben keine normalen 25-OH-Serumspiegel entwickelten, lasst jedoch eine weitere Optimierung des vorgestellten Behandlungskonzepts notwendig erscheinen.

Effekt von platinhaltigen Chemotherapien mit oder ohne Bevacizumab auf den Knochenstoffwechsel von Patientinnen mit primärem und platinsensitivem rekurrenten Ovarialkarzinom

Zielsetzung: Im Gegensatz zum Mammakarzinom liegen fur das epitheliale Ovarialkarzinom (EOC) kaum Erkenntnisse uber die Beeinflussung des Knochenstoffwechsels durch systemische Therapien vor. Im vorliegenden translationellen Projekt sollte der Einfluss platinhaltiger Chemotherapien (Ctx)± Bevacizumab (Bev) auf die Expression verschiedener Knochenstoffwechselparameter bei Patientinnen mit primarem bzw. platinsensitivem rekurrenten EOC naher untersucht werden. Methoden: 85 Patientinnen wurden eingeschlossen, 16 davon mit Ctx und Bev. Vor Therapiebeginn und vor jedem folgenden Ctx-Zyklus (C) von C1-C6 wurden folgende Serumparameter bestimmt: C-terminales Telopeptid des Typ I-Kollagens (ICTP) als Osteoklastenmarker, N-terminales Propeptid des Typ I-Kollagens (P1NP) als Osteoblastenmarker und alkalische Phosphatase (AP). Veranderungen der Knochenmarker unter Ctx wurden mittels Univarianzanalyse auf Signifikanz getestet. Ergebnisse: 350 Ctx Zyklen ohne und 87 Zyklen mit Bev wurden analysiert. Fur ICTP konnte ab C2 ein konstanter Abfall verzeichnet werden, Unter Ctx ohne Bev lag der Minimalwert bei 76%, bei Ctx mit Bev bei 56% des Basalwerts. Fur P1NP zeigte sich ein Abfall bereits nach dem ersten Zyklus mit einer Plateuabildung ab C3 bei Ctx ohne Ctx mit einem Minimalwert von 85% des Ausgangswerts. Bei Zugabe von Bev fiel P1NP wahrend des Gesamten Ctx-Verlauf auf 66% des Basalwertes ab. Bei Zugabe von Bev waren die negativen Effekte fur beide Parameter ausgepragter als bei Ctx alleine (ICTP: p = 0,043; P1NP: p = 0,002). Die AP lies waren des Therapieverlaufs keine signifikanten Veranderungen erkennen. Zusammenfassung: Eine platinbasierte Ctx beim EOC fuhrt zu einer signifikanten Inhibition der Osteoblasten- und (sekundar) der Osteoklastenaktivitat. Erstmals konnte gezeigt werden, dass Bev den Negativeffekt der Ctx auf den Knochenstoffwechsel signifikant verstarkt.