Detlev Latz

Radiotherapy for advanced adenoid cystic carcinoma: neutrons, photons or mixed beam?

PURPOSE To compare retrospectively radiotherapy with neutrons, photons, and a photon/neutron mixed beam in patients with advanced adenoid cystic carcinoma of the head and neck. Local control, survival, distant failure, and complications were analyzed. MATERIALS AND METHODS Between 1983 and 1995, 75 patients with inoperable, recurrent, or incompletely resected adenoid cystic carcinoma of the head and neck received radiotherapy that consisted of either fast 14.1 MV DT neutrons (median dose 16 neutron Gy), linac-based photon irradiation (median dose 64 photon Gy), or both (median dose 8 neutron Gy and 32 photon Gy). Follow-up ranged from 1 to 160 months (median 51 months), and the surviving patients had a minimum follow-up of 3 years at the time of analysis. RESULTS The actuarial 5-year local control was 75% for neutrons, and 32% for both mixed beam and photons (P = 0.015, log-rank). This advantage for neutrons in local control was not transferred to significant differences in survival (P > 0.1). The survival is dictated by the tumor diseases due to distant metastases occurring in 29 (39%) of the 75 patients. Positive lymph nodes were the only significant factor (P = 0.001) associated with the development of distant metastases although negative lymph nodes did not predict absence of distant metastases, but predicted a delay of occurrence. In multivariate analysis postoperative radiotherapy (P = 0.003) and small tumor size (P = 0.01) were associated with high local control, while primary therapy (P = 0.006) and negative lymph nodes (P = 0.01) were associated with longer survival. While acute toxicity was similar in all three radiotherapy groups, severe late grade 3 and 4 toxicity tended to be more prevalent (P > 0.1) with neutrons (19%) than with mixed beam (10%) and photons (4%). CONCLUSION Fast neutron radiotherapy provides higher local control rates than a mixed beam and photons in advanced, recurrent or not completely resected adenoid cystic carcinoma of the major and minor salivary glands. Neutron radiotherapy can be recommended in patients with bad prognosis with gross residual disease (R2), with unresectable tumors, or inoperable tumors. The type of radiation does not impact survival, which is dominated by the high number of distant metastases.

Open low-field magnetic resonance imaging in radiation therapy treatment planning

PURPOSE To evaluate the possibilities of an open low-field magnetic resonance imaging (MRI) scanner in external beam radiotherapy treatment (RT) planning. METHODS AND MATERIALS A custom-made flat tabletop was constructed for the open MR, which was compatible with standard therapy positioning devices. To assess and correct image distortion in low-field MRI, a custom-made phantom was constructed and a software algorithm was developed. A total of 243 patients (43 patients with non-small-cell lung cancer, 155 patients with prostate cancer, and 45 patients with brain tumors) received low-field MR imaging in addition to computed tomographic (CT) planning imaging between January 1998 and September 2001 before the start of the irradiation. RESULTS Open low-field MRI provided adequate images for RT planning in nearly 95% of the examined patients. The mean and the maximal distortions 15 cm around the isocenter were reduced from 2.5 mm to 0.9 mm and from 6.1 mm to 2.1 mm respectively. The MRI-assisted planning led to better discrimination of tumor extent in two-thirds of the patients and to an optimization in lung cancer RT planning in one-third of the patients. In prostate cancer planning, low-field MRI resulted in significant reduction (40%) of organ volume and clinical target volume (CTV) compared with CT and to a reduction of the mean percentage of rectal dose of 15%. In brain tumors, low-field MR image quality was superior compared with CT in 39/45 patients for planning purposes. CONCLUSIONS The data presented here show that low-field MRI is feasible in RT treatment planning when image correction regarding system-induced distortions is performed and by selecting MR imaging protocol parameters with the emphasis on adequate images for RT planning.

Moderate dose intraoperative and external beam radiotherapy for locally recurrent rectal carcinoma

BACKGROUND AND PURPOSE Late adverse effects (i.e. neuropathy, chronic bowel obstruction) limit the effective dose given in intraoperative radiotherapy (IORT) and external beam radiotherapy (EBRT). Initial results of a multi-modality treatment approach using moderate dose IORT and moderate dose EBRT are presented. PATIENTS AND METHODS Thirty-one consecutive patients with recurrent rectal carcinomas had IORT and EBRT after complete (R0, n = 14) or incomplete resection (R1, n = 9; R2, n = 8). The mean [ORT dose was 13.7 Gy (range 12-20 Gy) supplemented with an EBRT dose of 41.4 Gy. Twenty-two patients had preoperative EBRT and 22 patients had concomitant chemotherapy (5-FU, Leucovorine). RESULTS After a median follow-up of 28 months, 16 patients had re-recurrent disease and 11 patients had died. Nine patients failed locally (four in-field, four marginal and one anastomotic re-recurrence), three combined with distant metastasis, resulting in overall and IORT infield local control rates of 71% and 87%, respectively. Distant metastases alone were found in seven patients. The 4-year overall and relapse-free survival rates were 58% and 48%, respectively. After incomplete resection the local failure rate increased (R0 21%, R1/2 35%) and the 4-year relapse-free survival rate decreased significantly (29% versus 71%) due to a markedly increased distant metastasis rate (53% versus 7%). Acute and late toxicities were not increased. CONCLUSION The combination of moderate dose IORT and EBRT is a safe and efficacious component in a multi-modality treatment approach.

Effect and toxicity of endoluminal high-dose-rate (HDR) brachytherapy in centrally located tumors of the upper respiratory tract

Aim: To assess effect and toxicity of high-dose-rate afterloading (HDR) alone or in combination with external beam radiotherapy (EBRT) in centrally located tumors of the upper respiratory tract. Patients and Methods: From 1987 to 1996, 55 patients were treated. Twenty-one patients (group A1: 17 non-small-cell lung cancer [NSCLC], A2: 4 metastases from other malignancies) were treated using HDR alone due to a relapse after external beam irradiation. In 34 previously untreated and inoperable patients (group B1: 27 NSCLC, B2: 7 metastases from other malignancies) HDR was given as a boost after EBRT (30 to 60 Gy, median 50). HDR was carried out with a 192Ir source (370 GBq). The brachytherapy dose (group A: 5 to 27 Gy, median 20; B: 10 to 20 Gy, median 15) was prescribed to 1 cm distance from the source axis. A distanciable applicator was used in 39/55 patients. Results: In group A1, a response rate (CR, PR) of 53% (group B1: 77%) was reached. The median survival (Kaplan-Meier) was 5 months in group A1 (B1: 20 months). The 1-, 3- and 5-year local progression free survival rates (Kaplan-Meier) were 66% (15%), 52% (0%), and 37% (0%) in group B1 (group A1). Prognostic favorable factors in group B1 were a tumor diameter < 20 mm, the lack of radiological mediastinal involvement, a complete remission, and a Karnofsky performance status > 70. Grade-1 or 2 toxicity (RTOG/EORTC) occurred in 0% in group A and in 6% in group B. We observed no Grade-3 or 4 toxicity. Complications caused by persistent or progressive local disease occurred in 3 patients in group A (fatal hemorrhage, tracheomediastinal fistula, hemoptysis) and in 2 patients in group B (fatal hemorrhage, hemoptysis). Conclusions: HDR brachytherapy is an effective treatment with moderate side effects. In combination with external beam irradiation long-term remissions can be reached in one third of the patients.Ziel: Evaluierung von Effektivität und Toxizität der endoluminalen High-dose-rate-(HDR-)Brachytherapie als alleinige oder kombinierte (EBRT) Therapie bei zentral sitzenden Tumoren der oberen Atemwege. Patienten und Methode: Von 1987 bis 1996 wurden 55 Patienten behandelt. 21 Patienten (Gruppe A1: 17 Patienten mit nichtkleinzelligem Bronchialkarzinom [NSCLC], A2: vier Patienten mit Metastasen anderer Tumoren) wurden bei Lokalrezidiven nach vorheriger perkutaner Bestrahlung ausschließlich endoluminal bestrahlt. Bei 34 inoperablen und vorher unbehandelten Patienten (Gruppe B1: 27 NSCLC, B2: sieben Metastasen anderer Tumoren) wurde die Brachytheranie als Boost nach externer Bestrahlung (30 bis 60 Gy, Median 50) appliziert. Die endoluminale Bestrahlung wurde mit einer 192Ir-Quelle (370 GBq) durchgeführt. Dosiert wurde auf 1 cm Quellenabstand (Gruppe A: 5 bis 27 Gy, Median 20; B: 10 bis 20 Gy, Median 15). Ein distanzierbarer Spreizkorbapplikator wurde bei 39/55 Patienten verwendet. Ergebnisse: In Gruppe A1 wurde ein Therapieansprechen (CR, PR) in 53% erzielt (Gruppe B1: 77%). Das mediane Überleben (Kaplan-Meier) betrug fünf Monate in Gruppe A1 (B1: 20 Monate). Das lokalrezidivfreie Ein-, Drei- und Füf-Jahres-Überleben (Kaplan-Meier) betrug in Gruppe B1 ((A1) 66% (15%), 52% (0%) und 37% (0%). Als prognostisch günstige Faktoren konnten in Gruppe B1 ein Tumordurchmesser < 20 mm, radiologisch fehlende mediastinale Beteiligung, eine komplette Remission und ein Karnofsky-Index > 70 ermittelt werden. Grad-1- oder -2-Toxizität (RTOG/EORTC) trat in keinem Fall in Gruppe A und in 6% in Gruppe B auf. Wir beobachteten keine Grad-3- oder -4-Toxizität Tumorassoziierte Komplikationen kamen in drei Fällen in Gruppe A (Blutung, tracheomediastinale Fistelung, Hämoptysen) und in zwei Fällen in Gruppe B vor (Blutung, Hämoptysen). Schlußfolgerungen: Die endoluminale HDR-Brachytherapie ist eine effektive Therapie mit moderaten Nebenwirkungen. In Kombination mit externer Radiotherapie können Langzeitremissionen bei einem Drittel der Patienten erzielt werden.

Treatment of Primary Tracheal Carcinoma The Role of External and Endoluminal Radiotherapy

Background and Purpose: In a retrospective study the role of radiation therapy for the treatment of primary tracheal carcinoma was investigated. Patients and Methods: Between 1984 and 1997, 25 patients with primary tracheal carcinoma were treated with external beam radiotherapy (17 squamous-cell carcinoma [SCC], 8 adenoid cystic carcinoma [ACC], median dose SCC 60 Gy, ACC 55 Gy). An additional brachytherapy boost was carried out in 10/25 patients (median dose SCC 18 Gy, ACC 15 Gy). Ten patients underwent operative treatment. Results: The median survival (Kaplan-Meier) for patients with SCC was 33 months (ACC 94.2). The 1-, 2- and 5-year survival rates (Kaplan-Meier) for patients with SCC were 64.7% (ACC 85.7%), 64.7% (ACC 85.7%), and 26% (ACC 85.7%). Patients with ACC and patients with a complete remission after treatment had a significantly better survival probability (log rank test, p < 0.05). An excellent or good relief of clinical symptoms was achieved in 88% of the patients with SCC (ACC 88%). Eleven patients were locally controlled at last follow-up (SCC: 5/17; ACC: 6/8). Grade 1 to 2 toxicity (RTOG/EORTC) occurred in 12% (SCC: 2/17, ACC: 1/8) and Grade 3 to 4 toxicity in 8% (SCC: 0/17, ACC: 2/8) of the patients. Persistent or progressive local disease caused complications in 5 patients (fatal hemorrhage n = 2, esophagotracheal fistula n = 2, tracheal necrosis n = 1). Conclusion: Radiation therapy is an effective treatment for primary tracheal neoplasms. Surgery followed by adjuvant radiotherapy and primary radiotherapy in inoperable cases represent potentially curative treatment options. Prospective multicenter studies are needed to determine the optimal radiotherapeutic approach.Hintergrund und Zielsetzung: In einer retrospektiven Studie wurde der Stellenwert der Strahlentherapie im Behandlungskonzept des primären Trachealkarzinoms untersucht. Patienten und Methode: Zwischen 1984 und 1997 wurden 25 Patienten mit primären Trachealkarzinom perkutan bestrahlt (17 Plattenepithelkarzinome [SCC], acht adenoidzystische Karzinome [ACC], mediane Dosis SCC 60 Gy, ACC 55 Gy). Ein zusätzlicher Brachytherapie-Boost wurde in 10/25 Fällen appliziert (mediane Dosis SCC 18 Gy, ACC 15 Gy). Zehn Patienten waren operabel. Ergebnisse: Bei Patienten mit SCC betrug das mediane Überleben (Kaplan-Meier) 33 Monate (ACC 94,2). Die Ein-, Zwei- und Fünf-Jahres-überlebensraten (Kaplan-Meier) lagen in der SCC-Gruppe bei 64,7% (ACC 85,7%), 64,7% (ACC 85,7%) und 26% (ACC 85,7%). Patienten mit ACC und Patienten mit einer kompletten Remission zeigten eine signifikant bessere Überlebenswahrscheinlichkeit (Log-Rank-Test, p < 0,05). Eine ausgezeichnete oder gute Linderung der klinischen Symptomatik wurde in 88% der Fälle mit SCC erreicht (ACC 88%). Bei der letzten Wiedervorstellung waren elf Patienten lokal kontrolliert (SCC 5/17; ACC 6/8). Grad-1- oder -2-Toxizität (RTOG/EORTC) trat in 12% (SCC:2/17, ACC: 1/8) und Grad-3- bis -4-Toxizität in 8% (SCC: 0/17, ACC: 2/8) der Patienten auf. Ein Tumorprogreß bzw. -rezidiv führte in fünf Fällen zu Komplikationen (fatale Blutung n = 2, Ösophagotracheale Fistel n = 2, tracheale Nektose n = 1). Schlußfolgerung: Die Strahlentherapie ist eine effektive Methode in der Therapie trachealer Neoplasien. Einen potentiell kurativen Ansatz bieten die Tumorresektion mit adjuvanter Bestrahlung sowie die primäre Radiotherapie bei inoperablen Tumoren. Innerhalb prospektiver Multicenterstudien sollte das optimale radiotherapeutische Vorgehen festgelegt werden.

Combined effects of ionizing radiation and 4-hydroperoxyifosfamide in vitro

BACKGROUND AND PURPOSE Combined radiochemotherapy has gained increasing interest in clinical applications. The effects of combined exposure of ionizing radiation and 4-hydroperoxyifosfamide (4HOOIF) on cell survival were investigated in vitro. MATERIALS AND METHODS Clonogenic survival of log phase V79, Caski (squamous carcinoma), Widr (colon carcinoma) and MRI-221 cells (human melanoma) was determined after combined exposure to 4HOOIF and radiation. Measurement of cell survival for different cell cycle phases was performed after mitotic shake-off (V79) or appropriate intervals after serum stimulation of plateau phase cells (Widr). Control of cell cycle distribution was performed using flow cytometry. RESULTS In all cell lines tested, a combined exposure resulted in cell killing that was greater than for independent action. While this type of radiosensitization was of minor magnitude for log-phase cells or cells in G1 substantial radiosensitization was detected for S-phase cells with enhancement ratios (calculated from the respective mean inactivation doses) of up to 1.5. CONCLUSIONS The results demonstrate the interaction of 4HOOIF and radiation-induced cell damage with marked cell cycle specificity. Since the largest combination effect was observed for the most radioresistant S-phase cells, damage interaction could be mediated by an interference of 4HOOIF with the repair/fixation pathway of radiation-induced potentially lethal damage.

Treatment of primary squamous cell carcinoma of the trachea: the role of radiation therapy

Eleven patients with SCC of the trachea were treated (ten primarily, one postoperatively) with megavolt irradiation (four in combination with brachytherapy). A median survival of 31 and a median disease free survival of 7.5 months was observed. Factors favourable for survival were the achievement of complete remission, the absence of mediastinal lymphnode involvement, and the use of additional brachytherapy.

Radiochemotherapy with paclitaxel: Synchronization effects and the role of p53

PurposeWe have studied the interaction of paclitaxel (Taxol) and radiation in V79 cells and human lymphoblasts with special emphasis on cell cycle effects and the role of p53.Material and MethodsV79 cells in log- and plateau-phase and human lymphoblasts (p53wt TK6 and p53mut WTK1) were used. Paclitaxel was given for 2 hours. Survival was determined using clonogenic assays. Cell cycle analysis was done using DNA flow cytometry.ResultsIn V79 cells there was a dose dependent delay of colony formation after paclitaxel. The LD50 was about 0.4 μM with a 2-hour exposure. In exponentially growing cells, there was an accumulation of 40% of cells in G2/M 6 hours after paclitaxel. The dose modification factor was about 3.9 when radiation was given 6 hours after 0.3 μM paclitaxel for 2 hours. Synchronization experiments using serum starvation and induction showed that synchronization was not sufficient to induce a comparable dose modification factor. Human lymphoblasts with mutated p53 (WTK1, LD50=75 μM) were more resistant to paclitaxel than wild type p53 cells (TK6, LD50=25 μM).ConclusionThe radiosensitization induced by paclitaxel was critically dependent on the timing of irradiation and chemotherapy, although synchronization alone was not sufficient to explain the dose modification. Lymphoblasts with mutated p53 were less sensitive than wild type p53 cells.ZusammenfassungZielWir untersuchten die Interaktion von Paclitaxel (Taxol) und Bestrahlung in V79-Zellen und human en Lymphoblasten unter besonderer Berticksichtigung der Zellzykluseffekte und der Rolle von p53.Material und MethodikV79-Zellen in Exponential- und Plateauphase und humane Lymphoblasten (p53wt TK6 und p53mut WTK1) wurden benutzt. Paclitaxel wurde jeweils für zwei Stunden gegeben. Das zelluläre Überleben wurde mit klonogenen Assays bestimmt. Die Zellzyklusanalysen wurden mit der DNA-Flußzytometrie durchgeführt.ErgebnisseBei V79-Zellen wurde eine dosisabhängige Verzögerung der Koloniebildung beobachtet. Die LD50 lag nach einer Zwei-Stunden-Exposition bei 0,4 μM. Bei exponentiell wachsenden Zellen kam es zu einer Akkumulation von 40% der Zellen in der G2/M-Phase sechs Stunden nach Paclitaxel-Gabe. Der Dosismodifikationsfaktor lag bei 3,9, wenn sechs Stunden nach Applikation von 0,3 μM Paclitaxel bestrahlt wurde. Die Synchronisationsexperimente mit Serumentzug und -induktion zeigten, daß die Synchronisation allein nicht ausreichte, einen vergleichbaren Effekt zu erzielen. Die humanen Lymphoblasten mit mutiertem p53 (WTK1, LD50=75 μM) waren resistenter für Paclitaxel als die p53-Wildtypzellen (TK6, LD50=25 μM).SchlußfolgerungDie Radiosensibilisierung durch Paclitaxel hängt ganz wesentlich von der zeitlichen Abfolge der Bestrahlung und der Chemotherapie ab, obwohl die Synchronisation allein nicht ausreicht, urn den Dosismodifikationsfaktor zu erklären, Lymphoblasten mit mutiertem p53 waren weniger empfindlich als p53-Wildtypzellen.

MRT-simulation von bronchialkarzinomen mittels eines offenen niederfeld-MRT

AIM The initial target volume for primary radiation therapy of lung cancer is usually determined with the aid of computed tomography. Due to the axial CT-scans the simulation of the RT-field is often difficult. MRI in its superior ability to demonstrate and characterize soft tissue and its possibilities of multiplanar imaging can be beneficial. As MRI is less available and more expensive the use of MRI in radiotherapy planning is still restricted. With the introduction of open low-field MRI-systems there is now a cost-saving alternative. The aim of this study was the clinical evaluation of the use of a new open low-field MRI in radiotherapy planning of bronchogenic cancer. PATIENTS AND METHODS Fifteen patients undergoing primary radiotherapy for lung cancer were studied using an open low-field MRI-system (Picker Outlook 0.23 Tesla). Conventional CT-assisted treatment planning was compared to a MRI-assisted procedure. Contours from coronary T1-weighted MRI-sections were superimposed onto the simulator radiograph using a subtrascope (MR-simulation). RESULTS Open low-field MR-imaging using T1-weighted sequences resulted in excellent delineation of tumor masses from mediastinal fat, the airways and the vascular structures as well as the radial tumor infiltration into the vicinity of the lung (Figures 1a to 1c). This allowed an exact and reproducible transfer of tumor contours onto the simulator radiograph. The MR-simulation led to optimization in the field configuration in 5/15 patients (Figure 2). CONCLUSIONS Open low-field MRI-systems can be very useful in treatment planning. They are less expensive and need less extensive rebuilding compared to high-field MRI-systems. In the radiotherapy planning of bronchogenic carcinoma the MR-simulation is reasonable and clinically practicable. One of the main advantages of open MRI-systems in comparison to CT and standard MRI-systems in radiotherapy planning is that there is a much greater variety of treatment positions.HintergrundDas initiate Zielvolumen zur primären Strahlentherapie von Bronchialkarzinomen wird meist anhand der Tumorausdehnung im CT bestimmt. Die transversale Schnittführung erschwert hierbei die Umsetzung in die Simulation der Bestrahlungsfelder. Die MRT bietet durch den besseren Weichteilkontrast und die freie Wahl der Schnittebenen Vorteile. Mit der Entwicklung der Niederfeld-MRT-Systeme steht jetzt eine kostengünstige Alternative zur Verfügung. Ziel dieser Studie war die klinische Evaluierung eines offenen Niederfeld-MRT-Gerätes in der Therapiesimulation von nichtkleinzelligen Bronchialkarzinomen.Patienten und Methode15 Patienten mit primärer Strahlentherapie bei Bronchialkarzinom wurden mittels eines offenen Niederfeld-MRT (Picker Outlook, 0,23 T) untersucht. Die Fremd-CT-gestützte Simulatortechnik wurde mit einem MRT-gestützten Vorgehen verglichen. In Bestrahlungsposition angefertigte koronare native T1-gewichtete MRT-Schnitte wurden dazu subtraskopisch mit den Simulatoraufnahmen überlagert.ErgebnisseDie Niederfeld-MRT unter Verwendung von nativen T1-Sequenzen zeigte eine gute Abgrenzbarkeit des Tumorgewebes vom mediastinalen Fettgewebe und vom Gefäß- und Bronchialbaum sowie eine gute Darstellung der feinen Tumorausläufer ins Lungenparenchym. Die Wahl der Schnittführung in der Simulationsebene erlaubte eine topographisch exakte und gut reproduzierbare Übertragung des Tumors auf die Simulatoraufnahme. Insgesamt mußte bei 5/15 Patienten aufgrund der MRT-Simulation eine Optimierung der Bestrahlungsfelder durchgeführt werden.SchlußfolgerungOffene Niederfeld-MRT-Systeme können eine wertvolle Ergänzung für die Therapieplanung sein. Sie sind vergleichsweise kostengünstig. Bei der Bestrahlungsplanung von Bronchialkarzinomen erscheint die MRT-Simulation in speziellen Fällen sinnvoll, und sie ist klinisch anwendbar. Ein entscheidender Vorteil der offenen MRT gegenüber der CT und auch der geschlossenen MRT liegt in den freier wählbaren Lagerungsmöglichkeiten des Patienten.AbstractAimThe initial target volume for primary radiation therapy of lung cancer is usually determined with the aid of computed tomography. Due to the axial CT-scans the simulaton of the RT-field is often difficult. MRI in its superior ability to demonstrate and characterize soft tissue and its possibilities of multiplanar imaging can be beneficial. As MRI is less available and more expensive the use of MRI in radiotherapy planning is still restricted. With the introduction of open low-field MRI-systems there is now a cost-saving alternative. The aim of this study was the clinical evaluation of the use of a new open low-field MRI in radiotherapy planning of bronchogenic cancer.Patients and MethodsFifteen patients undergoing primary radiotherapy for lung cancer were studied using an open low-field MRI-system (Picker Outlook 0.23 Tesla). Conventional CT-assisted treatment planning was compared to a MRI-assisted procedure. Contours from coronary T1-weighted MRI-sections were superimposed onto the simulator radiograph using a subtrascope (MR-simulation).ResultsOpen low-field MR-imaging using T1-weighted sequences resulted in excellent delineation of tumor masses from mediastinal fat, the airways and the vascular structures as well as the radial tumor infiltration into the vicinity of the lung (Figures la to 1c). This allowed an exact and reproducible transfer of tumor contours onto the simulator radiograph. The MR-simulation led to optimization in the field configuration in 5/15 patients (Figure 2).ConclusionsOpen low-field MRI-systems can be very useful in treatment planning. They are less expensive and need less extensive rebuilding compared to high-field MRI-systems. In the radiotherapy planning of bronchogenic carcinoma the MR-simulation is reasonable and clinically practicable. One of the main advantages of open MRI-systems in comparison to CT and standard MRI-systems in radiotherapy planning is that there is a much greater variety of treatment positions.

Sensitivity of Neutral Filter Elution but Not PFGE Can Be Modified by Non-dsb Chromatin Damage

Hamster V79 fibroblast cells and human squamous carcinoma cells (Caski) were exposed to 60Co radiation and DNA double-strand break (dsb) induction was analysed by DNA elution at neutral pH from polycarbonate filter or out of an agarose matrix in pulsed-field electrophoresis (PFGE). While dsb yields were equal for the two cell lines (using 125-iodine calibration) a reduced responsiveness of filter elution was found for V79 versus Caski cells. This difference could be abolished when additional single-strand breaks (ssb) were introduced by an incubation at 10(-4) M H2O2 for up to 40 min that itself did not give a response in neutral elution. No such lack of specificity for the detection of dsb was seen in electrophoretic elution where also the influence of peroxide incubation was absent. The presumed potential of ssb to modify dsb detection was paralleled by the kinetics of dsb rejoining: a pronounced transient increase of DNA elution from filters was observed for V79 cells (less prominent with Caski cells) at 15-40 which is thought to reflect the occurrence of secondary ssb from incisions during base damage repair. Rejoining measured by PFGE did not show this behaviour. The results suggest that ssb may aid decondensation of the chromatin during lysis of cells required for an efficient release of dsb fragments when supported on filters, but which depends on cell type and is less critical in electrophoretic elution out of an agarose matrix. This involvement of ssb in the neutral filter elution assay appears to be contrary to published data obtained with different experimental systems. The finding of an increase of DNA elution from filters due to hyperthermia at 45 degrees C is also taken to indicate an involvement of non-dsb chromatin damage in the response of filter elution at neutral pH with V79 but not with Caski cells.