Focal laser ablation as clinical treatment of prostate cancer: report from a Delphi consensus project.

PURPOSE: To define the role of focal laser ablation (FLA) as clinical treatment of prostate cancer (PCa) using the Delphi consensus method. METHODS: A panel of international experts in the field of focal therapy (FT) in PCa conducted a collaborative consensus project using the Delphi method. Experts were invited to online questionnaires focusing on patient selection and treatment of PCa with FLA during four subsequent rounds. After each round, outcomes were displayed, and questionnaires were modified based on the comments provided by panelists. Results were finalized and discussed during face-to-face meetings. RESULTS: Thirty-seven experts agreed to participate, and consensus was achieved on 39/43 topics. Clinically significant PCa (csPCa) was defined as any volume Grade Group 2 [Gleason score (GS) 3+4]. Focal therapy was specified as treatment of all csPCa and can be considered primary treatment as an alternative to radical treatment in carefully selected patients. In patients with intermediate-risk PCa (GS 3+4) as well as patients with MRI-visible and biopsy-confirmed local recurrence, FLA is optimal for targeted ablation of a specific magnetic resonance imaging (MRI)-visible focus. However, FLA should not be applied to candidates for active surveillance and close follow-up is required. Suitability for FLA is based on tumor volume, location to vital structures, GS, MRI-visibility, and biopsy confirmation. CONCLUSION: Focal laser ablation is a promising technique for treatment of clinically localized PCa and should ideally be performed within approved clinical trials. So far, only few studies have reported on FLA and further validation with longer follow-up is mandatory before widespread clinical implementation is justified.

Repair of oxidative DNA damage by amino acids.

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

Enhancement of human tumor cell killing by L-canavanine in combination with gamma-radiation.

On the basis of several physiological properties of L-canavanine, we have tested the prediction that this analogue of arginine would enhance the cytotoxic effects of gamma-rays in mammalian cells. Using the human colonic tumor cell line, HT-29, time-dose studies were performed with log-phase cultures in order to determine conditions which maximize the incorporation of L-canavanine into cellular proteins while leaving a large fraction of the cells viable for subsequent gamma-ray survival measurements. At an input ratio of 2.5 (L-canavanine:arginine), the analogue exerted a cytostatic effect on the cells for at least 6 days following one cell division. Little cell killing (less than 20%) by clonogenicity was caused by L-canavanine during the first 12 hr of treatment of log-phase cells, even at a L-canavanine:arginine ratio of 20. A 24-hr exposure, however, produced an exponential decrease in survival as a function of L-canavanine concentration. The interaction between L-canavanine treatment and gamma-ray damage with respect to cell survival was examined under several conditions and times based on the above findings. Optimal enhancement of X-ray-induced cytotoxicity (assayed by loss of clonogenicity) was observed with a 48-hr exposure to the analogue at a L-canavanine:arginine ratio of 10. A marked increase in radiosensitivity was observed when L-canavanine was administered either before or after irradiation of the cells. In both protocols, enhancement was seen at all radiation doses. Together with our earlier findings showing the antitumor activity of L-canavanine in L1210 murine leukemia, these results suggest the potential usefulness of this amino acid analogue in the treatment of cancer.

Effects of inhibitors of DNA strand break repair on HeLa cell radiosensitivity.

The effects of three drugs (hydroxyurea, 1-beta-arabinofuranosylcytosine, and diamide) known to inhibit DNA synthesis on the repair of ionizing radiation-induced DNA single-strand breaks measured by alkaline elution and on cellular radiosensitivity were examined. Inhibition of repair was observed at 10(-2) M hydroxyurea, 10(-4) M 1-beta-D-arabinofuranosylcytosine, and 5 X 10(-5) M diamide, levels causing only 10% cell kill. While the mechanisms by which the drugs inhibit DNA synthesis differ, they are equally effective at inhibiting repair; without drug, cells, after a dose of 10 grays, repair 35% of DNA strand breaks in 3 min and a further 35% in 1 hr; with drug, only 10% is repaired in 3 min, and the deficiency in repair amount remains, even after 60 min. The effect of similar drug treatment on radiation-induced cell killing shows that radiosensitivity is increased; the major effect is reduction in D0 from 1.3 grays to approximately 0.8 grays with smaller effects on Dq. The data are consistent with the hypothesis that radiation produces potential double-strand breaks in DNA which, if not rapidly repaired, are converted into lethal actual double-strand breaks.

Uptake of WR-2721 derivatives by cells in culture: identification of the transported form of the drug.

When V79-171 cells are incubated in medium to which WR-1065 has been added the cells accumulate WR-1065 and disulfides of WR-1065 (WRSS) in a ratio of about 10:1. Analysis of the culture medium showed that it contained primarily WR-1065 but that significant levels of the symmetrical disulfide WR-33278 and of the mixed disulfide of WR-1065 with cysteine were also present. Since incubation of cells with either of the latter disulfides did not lead to uptake it was concluded that WR-1065 is the form of the drug taken up. The uptake rate on a per cell basis was shown to be independent of cell density, to be first order in the WR-1065 concentration in the incubation medium, to increase as [H+]-1.2 at medium pH values from pH 6.8 to 8.0, and to have a Q10 value (rate increase per 10 degrees C temperature increase) of 2.9 +/- 0.3 between 2 and 37 degrees C. Rates of WR-1065 uptake measured for HeLa, HT29/SP-1d, Me-180-VCII, Ovary 2008, and WI-38 cell lines were found to be similar to that measured for V79-171 cells. The results are consistent with uptake by nonmediated, passive diffusion of the uncharged form of WR-1065 across the plasma membrane but uptake mediated by a membrane transport system could not be rigorously excluded. Based upon these results and earlier findings it is postulated that the lower drug uptake seen in tumors as compared with normal tissues in animals treated with WR-2721 results from a combination of (a) slower conversion of WR-2721 to WR-1065 in tumors as a consequence of the lower inherent level of alkaline phosphatase and lower pH in tumors and (b) a decreased uptake rate of the WR-1065 present in tumors as a consequence of their lower pH.

Radioprotection of cells in culture by WR-2721 and derivatives: form of the drug responsible for protection.

Studies of V79-171 cells were undertaken to determine what extracellular or intracellular derivative of the drug WR-2721 is associated with radioprotection. The effect of preincubation at 23 +/- 1 degree C with WR-2721, and with derivatives of WR-2721 produced in medium containing alkaline phosphatase, upon survival of cells following subsequent gamma-irradiation was examined. It was established that WR-2721, WR-1065, WR-33278, WRSSCys, and other disulfide forms produced by reactions of WR-1065 with the medium do not provide significant protection when present only extracellularly. Protection was found to correlate with cellular levels of the thiol form of the drug (WR-1065) but not with the cellular level of the disulfide forms of WR-1065. Similar results were obtained with HeLa, Me-180, Ovary 2008, HT-29/SP-1d, and Colo 395 cell lines showing that human tumor cell lines behave in the same fashion as the V79-171 nontumorigenic hamster diploid cell line. None of the drug forms produced significant cytotoxicity under the conditions used. It was concluded that it is the cellular level of WR-1065 at the time of irradiation which determines protection. The results are consistent with protection mechanisms involving scavenging of hydroxyl radicals, hydrogen atom transfer to DNA radicals, depletion of oxygen near DNA, enhancement of rapid biochemical repair processes, or some combination of these mechanisms.

Integrating chemohormonal therapy and surgery in known or suspected lymph node metastatic prostate cancer.

BACKGROUND: Prostate cancer persisting in the primary site after systemic therapy may contribute to emergence of resistance and progression. We previously demonstrated molecular characteristics of lethal cancer in the prostatectomy specimens of patients presenting with lymph node metastasis after chemohormonal treatment. Here we report the post-treatment outcomes of these patients and assess whether a link exists between surgery and treatment-free/cancer-free survival. METHODS: Patients with either clinically detected lymph node metastasis or primaries at high risk for nodal dissemination were treated with androgen ablation and docetaxel. Those responding with PSA concentration <1 ng ml(-1) were recommended surgery 1 year from enrollment. ADT was withheld postoperatively. The rate of survival without biochemical progression 1 year after surgery was measured to screen for efficacy. RESULTS: Forty patients were enrolled and 39 were evaluable. Three patients (7.7%) declined surgery. Of the remaining 36, 4 patients experienced disease progression during treatment and 4 more did not reach PSA <1. Twenty-six patients (67%) completed surgery, and 13 (33%) were also progression-free 1 year postoperatively (8 with undetectable PSA). With a median follow-up of 61 months, time to treatment failure was 27 months in the patients undergoing surgery. The most frequent patterns of first disease recurrence were biochemical (10 patients) and systemic (5). CONCLUSIONS: Half of the patients undergoing surgery were off treatment and progression-free 1 year following completion of all therapy. These results suggest that integration of surgery is feasible and may be superior to systemic therapy alone for selected prostate cancer patients presenting with nodal metastasis.

Interpretation of statistical methodology associated with maintenance trials.

Maintenance trials in ulcer disease provide a scientific framework through which the prophylactic performances of two or more therapies can be compared with respect to ulcer recurrence. For this purpose, a primary source of data is the endoscopic classification of patients at protocol-scheduled visits as well as at unscheduled visits. Several statistical issues are relevant to the analysis of this endoscopic information. First, the extent to which the respective patients have different data patterns for their ulcer recurrence status must be taken into account by actuarial methods that adjust for the length of follow-up time. Second, the role of multiple investigators requires careful attention when information is combined from them. A third issue is concerned with the assessment of the variation of ulcer recurrence experience across subgroups with respect to patient demographics, medical history, and baseline characteristics. Statistical methods can effectively address these issues so that the results of maintenance studies can be interpreted through estimates for ulcer recurrence rates. In this way, they can meaningfully support clinical conclusions about the performance of alternative therapies for maintaining the absence of ulcer disease over time.

Mechanisms of DNA repair and their potential modification for radiotherapy.

The potentiation of radiation damage, which can be accomplished by the inhibition of repair, is estimated from published studies of repair deficient mutants. Sensitization factors as high as 10 have been achieved. Because it has previously been suggested that the most probable lethal lesion is a DNA double strand break (DSB), it is not surprising that cells deficient in repairing this type of damage are the most radiosensitive. The structures of DNA DSBs and other Locally Multiply Damaged Sites (LMDS) (involving both single strand breaks (SSB) and base damage sites) are reviewed, together with the processes by which cells may attempt to repair these lesions. Repair processes occur in competition with damage fixation, again, mechanisms of damage fixation are predicted from studies in model systems. A strategy for inhibiting the repair processes is devised that consists of holding the first SSB constituent of the LMDS open by repairing in the presence of deoxynucleoside analogues, such as ara-C, so that there is a higher probability of the formation of a DSB upon cleavage of the second site (on the other strand) by hydrolysis of a labile bond or by endonuclease cleavage of a base damaged site. To achieve preferential sensitization of tumor vs. normal tissue it may be possible to take advantage of the deficiency in alkaline phosphatase in tumor vs. normal vasculature, that is, in analogy with treatment with WR-2721. The deoxynucleoside analogue would be delivered together with the phosphate ester (deoxynucleotide) of the correct deoxynucleoside, for example, ara-C, in the presence of deoxycytidine monophosphate (dCMP). Higher alkaline phosphatase levels in normal tissue capillaries would hydrolyse the dCMP to deoxycytidine, which competes effectively with ara-C in repair replication.

Pulse radiolysis studies of WR-1065.

WR-1065, the dephosphorylated sulfhydryl form of WR-2721 has been suggested as the metabolite responsible for the radioprotective abilities of the latter and as being active during the radiation chemical stage of damage production. Hence this study was performed to determine some of the radiation chemical parameters of the compound. Pulse radiolysis techniques, developed for use with SH compounds, were employed in the current work: Three systems were used to attempt to measure the rate constant for reaction of OH. radicals with WR-1065; (a) Competition with phenylalanine, in which the decrease in the amount of RSSR- caused by the addition of phenylalanine is monitored; this failed because of the absence of an absorbance attributable to RSSR-. (b) Competition with 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), monitoring the decrease in OH. induced ABTS absorbance caused by WR-1065 addition. This was unsuccessful due to reaction of RS. with ABTS. (c) Competition with CNS-, observing the decrease in (CNS)-2 caused by WR-1065 addition indicates that the second order rate constant for reaction of OH. with WR-1065 is 9.2 +/- 0.3 X 10(9) M-1 s-1. To investigate the ability of WR-1065 to react with DNA radicals the yield of ABTS radicals was monitored in a situation where DNA scavenges 50% of the OH. radicals in the presence of ABTS (20%) and WR-1065 (30%). DNA radicals were shown not to react with ABTS but WR-1065 radicals do, the absence of additional ABTS absorbing species indicates that DNA radicals do not react with WR-1065 under the experimental conditions used. An attempt was made to investigate the possibility that WR-1065 is concentrated close to DNA macromolecules in solution: The rate constant for reaction Br2- with WR-1065 was measured in the presence and in the absence of excess DNA (with which Br2- was not observed to react). The observed rates of reaction were independent of the presence of DNA indicating that the latter does not affect the availability of WR-1065 for reaction with Br2- radical. Reaction of WR-1065 with Br2- was used as a probe for determining the pK of the SH group. The rate of reaction increases from 1.8 X 10(8) M-1 s-1 at pH 6.3 to 1.6 X 10(9) M-1 s-1 at pH 8.4, the mid-point of the increase indicates a pK of 7.3.

Radiation mutagenesis: the initial DNA lesions responsible.

This paper is an attempt to initiate a discussion about the causes of radiation-induced mutagenesis. It considers the evidence for the involvement of singly base-damaged sites as sources of this mutagenesis. Several sets of published data are examined: These include endogenous levels of oxidized bases and their production by natural oxidative processes, and yields of mutants produced by hydrogen peroxide and by high-LET radiation. These considerations lead to the conclusion that, at the levels at which they are produced by ionizing radiation, DNA base-damaged sites are not a cause of radiation-induced mutagenesis. In analogy with previous work we invoke the involvement of multiply damaged sites (such as double-strand breaks) produced by radiation as the causative lesions.

DNA damage as the cause of ionizing radiation-induced gene activation.

Tyrosine kinases have been shown to be activated by both UVA and UVC light and gamma irradiation. For UV exposure, a non-DNA target has been proposed. Here the possibility of a non-DNA target for ionizing radiation is examined. A comparison of the fluence/dose to which the cells were exposed to elicit the response indicates that three orders of magnitude greater numbers of potentially damaging events occur after UVC exposure than after gamma irradiation. Thus, while the involvement of a non-DNA target by UVC is possible, the probability of "hits" on such targets by ionizing radiation events is remote. The levels of intracellular endogenous oxidation damage are compared to those produced by ionizing radiation. This indicates that an oxidation product which is rapidly repaired and which is therefore present at low endogenous levels, such as the DNA single-strand break, is a candidate lesion for signal induction.

A new method for introducing double-strand breaks into cellular DNA.

A novel method is used to introduce double-strand breaks into cellular DNA containing controlled levels of 5-bromo-2'-deoxyuridine (BrdU). Chinese hamster V79 cells substituted with BrdU are treated with Hoechst dye #33258 and then exposed to UVA light. Using neutral elution (pH 7.2) the yield of DNA double-strand breaks is found to be linearly dependent on the level of BrdU substitution (0.36-7.5%), concentration of Hoechst dye (0-100 micrograms cm-3), and fluence of UVA light (0.2-8 kJ m-2). The yield of double-strand breaks produced by this photolysis treatment is 5.1 x 10(-6) breaks/BrdU residue/kJ m-2, regardless of whether one or both strands of the DNA polymer contain BrdU. No double-strand breaks are detected in the absence of Hoechst dye, BrdU, or UVA light. The formation of double-strand breaks appears to involve strand cleavage at a BrdU site on one strand with cleavage in the opposite strand not necessarily requiring the presence of BrdU. The utility of this photolytic regimen in modeling the biological significance of double-strand break lesions and some putative mechanisms for their formation are discussed.

Response of bromodeoxyuridine-substituted Chinese hamster cells to UVA light exposure in the presence of Hoechst dye #33258: survival and DNA repair studies.

Previous work has established that DNA double-strand breaks (DSBs) are formed when Chinese hamster cells are substituted with 5-bromo-2'-deoxyuridine (BrdU) and exposed to UVA light in the presence of Hoechst dye #33258. Double-strand breaks produced by this treatment (5.1 x 10(-6) DSBs/BrdU residue/kJ m-2) were found to depend linearly on the level of BrdU substitution, Hoechst dye and fluence of UVA light. To examine the biological consequences of these novel DSBs, clonogenic assays were used to score cell survival, and elution assays were used to measure strand break levels at various times after photolysis. Using this system, marked cell killing was observed; photosensitivity could be increased by four orders of magnitude compared to cells without BrdU and dye. Decreases in the F0 value and the shoulder of survival curves followed increasing levels of BrdU substitution. In addition, the results indicate that DSBs produced by this photolysis protocol are two to three times more effective in causing cell killing than the DSBs produced by the action of ionizing radiation. To investigate the cause of the toxicity, repair of DSBs after photolysis was measured. Unexpectedly, DSB levels increased two- to threefold over 1 h at 37 degrees C, then decreased to initial damage levels over the next 2 h. The implications of this break induction are discussed in terms of mechanism and cell killing.

Radiosensitization of Chinese hamster V79 cells by bromodeoxyuridine substitution of thymidine: enhancement of radiation-induced toxicity and DNA strand break production by monofilar and bifilar substitution.

Bromodeoxyuridine (BrdU) competes with thymidine (TdR) for incorporation into DNA of exponentially growing V79-171 cells. Such cells show an enhancement of the radiation response as determined by clonogenic survival and DNA damage measured by filter elution techniques after doses up to 15 Gy. The degree of radiosensitization for both survival and rates of alkaline and neutral elution are dependent on percentage BrdU substitution and independent of whether BrdU is in one strand only (monofilar) or both strands (bifilar) of the DNA duplex: e.g., for 16% BrdU substitution distributed either monofilarly or partially bifilarly, there is an enhancement factor for Do of 1.55. At this percentage substitution, the enhancement factor for the rate of alkaline elution is 1.75 and that for the rate of neutral elution is 1.54. The greater the percentage BrdU substitution, the larger was the enhancement ratio for survival and radiation-induced strand breaks in both monofilarly and bifilarly substituted cells. The increase in cell radiosensitivity caused by BrdU substitution shows a better correlation with the increase in radiation-induced double-strand breaks than with the increase in radiation-induced single-strand breaks.

Yield of single-strand breaks due to attack on DNA by scavenger-derived radicals.

We have measured the yield of single-strand breaks (SSBs) in plasmid DNA after 137Cs gamma irradiation, in the presence of dimethyl sulfoxide (DMSO). In the presence of oxygen, the formation of SSBs is due to hydroxyl radical attack. As the DMSO concentration is increased from 10(-4) mol dm-3 to 1 mol dm-3, the SSB yield in the presence and absence of oxygen decreases by over 100-fold and less than 10-fold, respectively. From the DMSO and DNA concentration dependencies of the SSB yield in the absence of oxygen, the second-order rate constant for the reaction of the methyl radical (derived from DMSO) and DNA can be estimated as k2 = 8.8 x 10(4) dm3 mol-1 s-1. Several other scavengers were compared with DMSO under anoxia. Radicals derived from isopropyl alcohol and glycerol also caused SSB formation in DNA, while those from 2-deoxyribose, thymine, 1,3-dimethylthymine and 1,3-dimethyluracil did not. In the case of the scavenger tert-butyl alcohol, it is unclear whether the hydrogen atom (H.) or an organic radical is responsible for the higher SSB yield under anoxic conditions.

Effects of postirradiation temperature on the yields of radiation-induced single- and double-strand breakage in SV40 DNA.

The effects of postirradiation holding temperature on the yields of radiation-induced single- and double-strand breaks (SSBs and DSBs) in SV40 DNA have been measured by agarose gel electrophoresis. When the DNA is held at low temperatures (< or = 2 degrees C) before and during electrophoresis, the measured yields of radiation-induced SSBs and DSBs are twofold less than in samples exposed to room temperature. In contrast, if the DNA is incubated at 37 degrees C overnight, the yield of DSBs increases twofold over the room temperature assay, while the SSB yield increases only to a small extent (< or = 20%). From a comparison of the various yields, we suggest that low temperature stabilizes radiation-induced labile sites, and that the increased yield of DSBs at 37 degrees C is due either to the recruitment of spatially separate SSBs as DSBs by duplex melting, or to labile sites generating DSBs. The different routes to DSB formation are kinetically distinct. We conclude that room-temperature electrophoresis measures all SSBs including those from labile sites.

Variation of single-strand break yield with scavenger concentration for plasmid DNA irradiated in aqueous solution.

We have measured the yield of single-strand breaks (SSBs), induced by 137Cs gamma radiation as assayed by agarose gel electrophoresis, for three plasmids and SV40 DNA irradiated in aerobic aqueous solution. DNA SSBs are caused mainly by the hydroxyl radical under these conditions. To characterize the reactivity of DNA with the hydroxyl radical, we investigated the variation of the G value for SSBs [G(SSB)] with the concentration of added hydroxyl radical scavengers. We find that simple competition kinetics does not describe our results, but that a nonhomogeneous kinetics model is in good agreement. At a DNA concentration of 50 micrograms cm-3, G(SSB) for the direct effect is about 1 x 10(-5) mumol J-1 for the DNA substrates studied. This is equivalent to 2 x 10(-10) SSB Gy-1 Da-1. Estimates of the efficiency of SSB induction per OH. radical interaction with DNA (0.32-0.44) reveal that all plasmids are essentially equal in reactivity.

Variation of single-strand break yield with scavenger concentration for the SV40 minichromosome irradiated in aqueous solution.

We have measured the yield of single-strand breaks (SSBs) induced in aerobic aqueous solution by 137Cs gamma irradiation for the SV40 minichromosome as measured by agarose gel electrophoresis. Under these conditions, DNA SSBs are caused mainly by the hydroxyl radical. To characterize the reactivity of the SV40 minichromosome with the hydroxyl radical and to compare its behavior with that of naked DNA, we examined the variation of the G value for SSB formation, G(SSB), with the concentration of added hydroxyl radical scavengers. We find that simple competition kinetics is not applicable, but that a nonhomogeneous kinetics model is in much better agreement. Estimates of the efficiency of SSB induction per OH radical interaction with the SV40 minichromosome (0.04-0.05) indicate that this substrate is about five times more radioresistant than naked DNA at scavenging capacities < 10(8) s-1. At a DNA concentration of 50 micrograms ml-1, G(SSB) for the direct effect in the minichromosome is about 1 x 10(-5) mumol J-1 (2 x 10(-10) SSB Gy-1 Da-1), essentially equal to that for naked DNA.

The effect of superhelical density on the yield of single-strand breaks in gamma-irradiated plasmid DNA.

Using agarose gel electrophoresis, the formation of DNA single-strand breaks (SSBs) by 137Cs gamma irradiation was quantified in negatively supercoiled topological isomers of plasmid pUC18. The G value for SSB formation falls slightly from 1 x 10(8) to 8 x 10(-9) SSB Gy-1 Da-1 as the superhelical density varies from 0.00 to -0.08. This result is not in agreement with recent observations by others which suggest that increasing the negative superhelical density of plasmid DNA increases its sensitivity to X irradiation.