Heinz Schmidberger

Experimental studies on the role of antibody fragments in cancer radio-immunotherapy: Influence of radiation dose and dose rate on toxicity and anti-tumor efficacy

Whereas bivalent fragments have been widely used for radio‐immunotherapy, no systematic study has been published on the therapeutic performance of monovalent conjugates in vivo. The aim of our study was, therefore, to determine the therapeutic performance of 131I‐labeled Fab as compared to bivalent conjugates and to analyze factors that influence dose‐limiting organ toxicity and anti‐tumor efficacy. The maximum tolerated doses (MTDs) and dose‐limiting organ toxicities of the 131I‐labeled anti‐CEA antibody MN‐14 [IgG, F(ab′)2 and Fab] were determined in nude mice bearing s.c. human colon cancer xenografts. Mice were treated with or without bone marrow transplantation (BMT) or inhibition of the renal accretion of antibody fragments by D‐lysine or combinations thereof. Toxicity and tumor growth were monitored. Radiation dosimetry was calculated from biodistribution data. With all 3 131I‐labeled immunoconjugates [IgG, F(ab′)2 and Fab], the red marrow was the only dose‐limiting organ; MTDs were 260 μCi for IgG, 1,200 μCi for F(ab′)2 and 3 mCi for Fab, corresponding to blood doses of 17 Gy, 9 Gy and 4 Gy, respectively. However, initial dose rates were 10 times higher with Fab as compared to IgG and 3 times higher as compared to F(ab′)2. The MTD of all 3 immunoconjugates was increased by BMT by approximately 30%. In accordance with renal doses below 10 Gy, no signs of nephrotoxicity were observed. Despite lower absorbed tumor doses, at equitoxic dosing, Fab fragments were more effective at controlling tumor growth than the respective bivalent fragment or IgG, probably due to higher intratumoral dose rates. Our data indicate that the improved anti‐tumor effectiveness of antibody fragments as compared to IgG and the higher myelotoxicity at comparably lower red marrow doses are most likely due to the higher initial dose rates observed with antibody fragments. Int. J. Cancer 77:787–795, 1998.

Effect of Pentoxifylline and Tocopherol on Radiation Proctitis/Enteritis

Background and Purpose:Chronic radiation proctitis/enteritis is a relevant complication of pelvic irradiation, which is still mainly treated by supportive measures only. There is some evidence that the combined treatment with pentoxifylline and tocopherol might alter the pathogenesis of radiation-induced fibrosis. In a retrospective analysis the clinical benefit of the treatment with pentoxifylline/tocopherol on radiation-induced proctitis/enteritis was evaluated, compared to supportive care only.Patients and Methods:Of 30 patients with radiation-induced proctitis/enteritis grade I–II according to the RTOG/EORTC toxicity criteria, 21 were treated with pentoxifylline and tocopherol. Depending on physician’s decision nine patients received symptomatic treatment only.Results:With pentoxifylline/tocopherol treatment 15/21 patients (71%) experienced a relief of their symptoms. A reduction from grade I/II to grade 0 toxicity was observed in seven and from grade II to grade I toxicity in eight patients. No improvement was seen in six patients. The median time to improvement with pentoxifylline and tocopherol treatment was 28 weeks. In three of nine patients who were treated supportively only, deterioration of symptoms occurred. Three patients experienced no amelioration, and three patients with grade I toxicity experienced a spontaneous relief of their symptoms (33%).Conclusion:The combination treatment with pentoxifylline and tocopherol seems to have a benefit in patients with grade I–II radiation-induced proctitis/enteritis. The optimal schedule of treatment duration is not yet clear. From the observations made in this study it is assumed the treatment should be given for 6–12 months at least. A prospective phase II study should be undertaken to evaluate optimal treatment duration.Hintergrund und Ziel:Die strahleninduzierte chronische Proktitis/Enteritis ist eine relevante Komplikation nach Beckenbestrahlungen, die hauptsächlich symptomatisch therapiert wird. Die kombinierte Behandlung mit Pentoxifyllin und Tocopherol könnte die Pathogenese der strahleninduzierten Fibrose beeinflussen. In einer retrospektiven Analyse wurde der klinische Nutzen dieser Kombinationstherapie bei strahleninduzierter Proktitis/Enteritis ausgewertet und mit alleiniger symptomatischer Behandlung verglichen.Patienten und Methodik:Von 30 Patienten mit einer strahleninduzierten Proktitis/Enteritis Grad I–II nach den Kriterien der RTOG/EORTC wurden 21 mit Pentoxifyllin und Tocopherol behandelt. In Abhängigkeit von der ärztlichen Entscheidung wurden neun Patienten nur symptomatisch behandelt (Tabelle 1).Ergebnisse:15/21 Patienten (71%) unter Therapie mit Pentoxifyllin und Tocopherol erlebten eine Verbesserung ihrer Symptome (Tabelle 3, Abbildung 1). Eine Reduktion von Grad I/II zu Grad 0 trat bei sieben, von Grad II zu I bei acht Patienten auf. Keine Verbesserung konnte bei sechs Patienten erreicht werden. Die mittlere Zeit bis zur Verbesserung der Symptome unter Therapie mit Pentoxifyllin und Tocopherol betrug 28 Wochen (7 Monate) (Abbildung 3). Drei der neun symptomatisch therapierten Patienten erlitten eine Symptomverschlechterung. Drei Patienten erlebten keine Befundänderung, und drei Patienten mit Grad-I-Toxizität erfuhren eine spontane Verbesserung ihrer Symptome (33%) (Tabelle 4).Schlussfolgerung:Die Kombinationstherapie aus Pentoxifyllin und Tocopherol scheint einen Nutzen bei der strahleninduzierten Proktitis/Enteritis Grad I–II zu haben (Abbildung 2). Die optimale Behandlungsdauer ist nicht bekannt. Nach den Ergebnissen dieser Studie ist anzunehmen, dass die Medikamente mindestens 6–12 Monate gegeben werden sollten. Eine prospektive Phase- II-Studie sollte durchgeführt werden, um die optimale Behandlungsdauer zu evaluieren.

Therapeutic efficacy and dose-limiting toxicity of auger-electron vs. beta emitters in radioimmunotherapy with internalizing antibodies: Evaluation of 125I- vs. 131I-labeled CO17-1A in a human colorectal cancer model

Recent clinical results suggest that higher anti‐tumor efficacy may be achieved with internalizing monoclonal antibodies (MAbs) at lower toxicity when labeled with Auger‐electron, as compared to conventional β‐emitters. The aim of our study was to compare the toxicity and anti‐tumor efficacy of the 125I‐labeled internalizing MAb, CO17‐1A, with its 131I‐labeled form in a human colon cancer model in nude mice. Biodistribution studies were performed in nude mice bearing s.c. human colon cancer xenografts. For therapy, the mice were injected either with unlabeled 125I‐ or 131I‐labeled CO17‐1A at equitoxic doses. Control groups were left untreated, were given a radiolabeled isotype‐matched irrelevant antibody or a tumor‐specific, but noninternalizing antibody. The maximum tolerated activities (MTD) of 131I‐ and 125I‐CO17‐1A without artificial support were 300 μCi and 3 mCi, respectively. Myelotoxicity was dose‐limiting; bone marrow transplantation allowed for an increase of the MTD to 400 μCi of 131I‐17‐1A, whereas the MTD of 125I‐17‐1A with bone marrow support had not been reached at 5 mCi. Whereas no significant therapeutic effects were seen with unlabeled CO17‐1A, tumor growth was retarded with 131I‐CO17‐1A. With the 125I‐label, however, therapeutic results were clearly superior. In contrast, no significant difference was observed in the therapeutic efficacy of the 131I‐ vs. 125I‐labeled, noninternalizing antibodies. Our data indicate a superiority of Auger‐electron emitters, such as 125I, as compared to therapy with conventional β‐emitters with internalizing antibodies. The lower toxicity of Auger emitters may be due to the short path length of their low‐energy electrons, which can reach the nuclear DNA only if the antibody is internalized (as is the case in antigen‐expressing tumor tissue, but not in the stem cells of the red marrow). Int. J. Cancer 76:738–748, 1998.© 1998 Wiley‐Liss, Inc.

Radiotherapy for stages I and IIA/B testicular seminoma

Radiotherapy is generally accepted as a standard treatment for early‐stage testicular seminoma. Relapse rates of 2% to 5% in clinical stage I and 10% to 20% in stage IIA/B (according to the Royal Marsden classification) can be achieved. Disease‐specific survival reaches 100%. With such excellent cure rates, treatment‐related side effects gain particular importance. Therefore, a prospective multicenter trial was initiated for radiotherapy of testicular seminoma with limited treatment portals and low total doses of irradiation. In clinical stage I, 483 patients were treated with 26 Gy to the para‐aortic region only. In stage IIA, 42 patients and, in stage IIB, 18 patients received irradiation to the para‐aortic and high iliac lymph nodes with 30 and 36 Gy, respectively. With a median time to follow‐up of 55 months for stage I and 55.5 months for stage IIA/B, there were 18 (3.7 %) and 4 (6.7 %) cases of relapse in both treatment groups. Disease‐specific survival was 99.6% in stage I and 100% in stage IIA/B. Acute toxicity was dominated by moderate gastro‐intestinal side effects. No major late toxicity has been observed to date. Limited volume pure para‐aortic treatment for stage I and para‐aortic/high iliac irradiation for stage IIA/B with 26, 30 and 36 Gy, respectively, yields excellent cure rates with only moderate acute toxicity and is therefore recommended as standard treatment. Int. J. Cancer 83:823–827, 1999.

Radiotherapy and concomitant weekly 1-hour infusion of paclitaxel in the treatment of head and neck cancer—Results from a phase I trial

PURPOSEnTo define the maximum tolerated dose (MTD) by describing the dose-limiting toxicity (DLT) of weekly paclitaxel (PAC) given as a 1-h I.V. infusion in patients with head and neck cancer concomitant to irradiation.nnnMETHODS AND MATERIALSnPatients with unresectable or incompletely resected head and neck cancer were enrolled into a prospective, dose-escalating Phase I study. Toxicity was graded according to the WHO toxicity score. MTD dose was defined when two out of six patients developed DLT. The starting dose of PAC was 20 mg/m2 once weekly I.V. over 60 min, with a subsequent dose escalation of 10 mg/m2 in cohorts of three new patients. Radiation therapy was administered in three field technique over 6-7 weeks in 2.0 Gy/daily fractions for 5 consecutive days/week up to total doses of 60-70 Gy.nnnRESULTSnFrom 1994-1996, 18 patients completing three dose levels were included into the study. Altogether, 101 courses of chemotherapy were evaluable for toxicity. On the second dose level (30 mg/m2) one of three patients experienced DLT with Grade IV mucositis. On the next dose level with 40 mg/m2 PAC weekly one patient experienced DLT being prolonged Grade III mucositis. From the following three patients required, two patients showed no DLT. The third patient showed mucositis of WHO Grade 4 and died from hemorrhage caused by a rupture of the a pharyngeal wall. Dose level 2 (30 mg/m2) was repeated and one of the three newly treated patients again suffered from mucositis WHO Grade 4.nnnCONCLUSIONnWhen PAC is given weekly as a 1-h infusion concomitant to radiotherapy, MTD is 30 mg/m2 with mucositis being DLT; hematological and further nonhematological toxicity is mild.

Role of DNA-PK in the process of aberration formation as studied in irradiated human glioblastoma cell lines M059K and M059J.

Purpose : The participation of various DNA double-strand break repair mechanisms in the formation of chromosome aberrations is not yet fully understood. To investigate particularly the role of non-homologous end-joining, we analysed the formation of radiation-induced aberrations in a DNA-protein kinase (PK CS) -proficient cell line M059K and in a deficient cell line M059J. Materials and methods : Plateau-phase M059K and M059J cells were irradiated with low doses of X-rays. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations and total visible complex exchanges (complex aberrations) for chromosomes 4, 7 and 11 using the FISH method. M059K cells were also analysed in the presence of 50 µ;m wortmannin, a DNA-PK inhibitor. Results : DNA-PK-deficient cells showed a higher yield of simple stable and unstable and of complex aberrations in comparison with DNA-PK-proficient cells. The largest differences were observed for excess acentric fragments and for complex aberrations. DNA-PK inhibition by wortmannin in M059-K cells resulted in increased aberration yield in the same qualitative and quantitative manner as in M059-J cells. Conclusion : The results obtained with DNA-PK-deficient M059J cells and with DNA-PK-proficient M059K cells treated with wortmannin, an inhibitor of DNA-PK and ATM, suggest that the elimination of DNA-PK-dependent non-homologous end-joining can recruit a slow, error-prone repair process, which is DNA-PK independent and favours the increased formation of chromosome aberrations. The nature of this pathway and the way of its participation in aberration formation need further elucidation.

Effects of docetaxel in combination with radiation on human head and neck cancer cells (ZMK-1) and cervical squamous cell carcinoma cells (CaSki ).

The purpose of this study was to determine, as we did for paclitaxel, the cytotoxic and radiosensitizing potential of docetaxel in human head and neck cancer cells (ZMK‐1), and in cervical squamous cell carcinoma cells (CaSki). ZMK‐1 cells were incubated with docetaxel for 3, 9 or 24 hr before irradiation and 24 hr after irradiation. CaSki cells were incubated with docetaxel 24 hr before and after irradiation. For ZMK‐1 cells, the docetaxel concentrations (0.7, 0.7 and 0.35 nM) were determined to obtain approximately equivalent cell survival at the different incubation times (3, 9 and 24 hr, respectively). For CaSki cells, the necessary concentration of docetaxel was 0.07 nM. Radiation doses were given from 0 to 7 Gy. Cell survival was measured by a standard clonogenic assay after a 9‐day incubation. Flow cytometry was used to measure the capacity of docetaxel to accumulate cells in the G2/M phase of the cell cycle. We observed a weak accumulation of cells in the G2/M phase for the ZMK‐1 cells and a pronounced accumulation for CaSki cells. For docetaxel incubation before irradiation, the isoeffect enhancement ratios for ZMK‐1 cells determined at the 37% survival level were 1.18, 2.01, and 2.40 for pre‐incubation at 3, 9 and 24 hr, respectively; for CaSki cells the ratio was 1.44. For a docetaxel incubation of 24 hr after irradiation, the isoeffect enhancement ratios determined at the 37% survival level were 1.54 and 1.17 for the ZMK‐1, and CaSki cells, respectively. A radiosensitizing effect of docetaxel could be demonstrated unambiguously in the two cell lines used. In contrast to our previously published results with paclitaxel, docetaxel seems to be a better radiosensitizer than paclitaxel.

Effect of keratinocyte growth factor on the proliferation, clonogenic capacity and colony size of human epithelial tumour cells in vitro.

Purpose : The effect of recombinant human keratinocyte growth factor (rHuKGF) on the proliferation, clonogenic capacity and colony size of low-passage human epithelial tumour cells was tested in vitro. Materials and methods : Five tumour cell cultures derived from head and neck squamous cell carcinomas, three cultures derived from pleural effusions of carcinomas of different origin and normal human nasal epithelial cells were analysed in passages 2-4. Expression of FGF7 and its receptor (FGFR2) were determined by the RNase protection assay. Cells were incubated with rHuKGF (10-200 ng ml −1) 3 days before or immediately after plating for clonal growth in serum-depleted media. To determine cellular radiosensitivity, single doses of 1-8 Gy X-rays were applied. Colony formation as well as colony size, reflecting the number of cell divisions, was determined after 10-15 days of growth in rHuKGF-treated and control cells. Results : Normal nasal epithelial cells showed a two- to threefold increase in the number of cell divisions due to rHuKGF-treatment. In tumour cell cultures, significant stimulation of proliferation occurred in only one of eight samples. Tumour cells expressed FGF7 mRNA and protein, and low levels of FGFR2 mRNA. The addition of rHuKGF to the medium of the tumour cell cultures influenced neither radiation-induced impairment of proliferation nor clonogenic cell survival. Conclusion : rHuKGF has been shown to ameliorate the radiation tolerance of normal epithelia. The minimum in vitro tumour cell response to rHuKGF compared with normal epithelial cells suggests a potential for selective protection of normal epithelia during radiotherapy. The low FGFR2 expression as well as the FGF7 expression in the tumour cells may contribute to their resistance to rHuKGF treatment.

In vitro response of human dermal fibroblasts to X-irradiation: relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation.

Purpose : To analyse the relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation. Materials and methods : Plateau-phase human dermal fibroblasts from 18 donors were irradiated with graded doses of 1-6 Gy 200kV X-rays. Cell survival was determined by a colony-forming assay. Markers of differentiation or senescence were: spontaneous and radiation-induced clonal differentiation, which was determined morphologically and by the cellular potential to proliferate in clonal culture, also single-cell g -galactosidase (g -gal) staining at pH 6.0; and the secretion of transforming growth factor- g (TGF- g 1) into the culture medium. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and the excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations for chromosomes 4, 7 and 9 using the FISH method. Results : A broad spread was found in the shapes of the survival curves, with SF2 ranging from 0.041 - 0.015 to 0.63 - 0.05. Radiation-induced clonal differentiation as well as the secretion of TGF- g 1 was elevated in radiosensitive samples. With respect to chromosome aberrations, a significant correlation was found between clonogenic survival and radiation-induced excess acentric fragments. Conclusions : In the fibroblast cell system, in vitro radiosensitivity is determined not only by processes directly involved in DNA-damage recognition and repair, but also by intracellular signalling cascades, which will lead to differentiation processes.

The combined effect of interferon beta and radiation on five human tumor cell lines and embryonal lung fibroblasts

PURPOSEnThe combined effect of natural Interferon-beta (n-IFN-beta) and ionizing radiation was tested in vitro on 5 different tumor cell lines and 1 embryonal lung fibroblast cell line.nnnMATERIALS AND METHODSnThe following cell lines were used: A549 (lung cancer), MCF-7 (breast cancer), CaSki (cervical cancer), WiDr (colon cancer), ZMK-1 (head and neck cancer), and MRC-5 (embryonal lung fibroblast line). Cells were incubated with n-IFN-beta (30 I.U./ml to 3000 I.U./ml) 24 h before irradiation. Irradiation was given as single dose between 1 and 6 Gy. Cell survival was evaluated using a standard colony-forming assay.nnnRESULTSnIncubation with n-IFN-beta enhanced the effect of radiation in all tumor cell lines tested. The maximum sensitizing enhancement ratios (SER) at the 37% survival level were: 1.66 for A549 cells, 1.47 for CaSki cells, 1.56 for MCF-7 cells, 1.40 for WiDr cells, and 1.57 for ZMK-1 cells. In the nonneoplastic MRC-5 cell line, no radiosensitizing effect of n-IFN-beta could be demonstrated. The linear quadratic fit of the survival curves showed an increase of the alpha-component for all tumor cell lines treated with n-IFN-beta.nnnCONCLUSIONSnIFN-beta enhanced the effect of radiation in the tumor cell lines, but not in the nonmalignant lung fibroblasts. The increase of the alpha component in the survival curves indicates that impaired radiation repair or the accumulation of sublethal damage might play a role for the radiosensitizing effect of n-IFN-beta.