Consolidative proton therapy after chemotherapy for patients with Hodgkin lymphoma.

BACKGROUND: We investigated early outcomes for patients receiving chemotherapy followed by consolidative proton therapy (PT) for the treatment of Hodgkin lymphoma (HL). PATIENTS AND METHODS: From June 2008 through August 2015, 138 patients with HL enrolled on either IRB-approved outcomes tracking protocols or registry studies received consolidative PT. Patients were excluded due to relapsed or refractory disease. Involved-site radiotherapy field designs were used for all patients. Pediatric patients received a median dose of 21 Gy(RBE) [range 15-36 Gy(RBE)]; adult patients received a median dose of 30.6 Gy(RBE) [range, 20-45 Gy(RBE)]. Patients receiving PT were young (median age, 20 years; range 6-57). Overall, 42% were pediatric (≤18 years) and 93% were under the age of 40 years. Thirty-eight percent of patients were male and 62% female. Stage distribution included 73% with I/II and 27% with III/IV disease. Patients predominantly had mediastinal involvement (96%) and bulky disease (57%), whereas 37% had B symptoms. The median follow-up was 32 months (range, 5-92 months). RESULTS: The 3-year relapse-free survival rate was 92% for all patients; it was 96% for adults and 87% for pediatric patients (P = 0.18). When evaluated by positron emission tomography/computed tomography scan response at the end of chemotherapy, patients with a partial response had worse 3-year progression-free survival compared with other patients (78% versus 94%; P = 0.0034). No grade 3 radiation-related toxicities have occurred to date. CONCLUSION: Consolidative PT following standard chemotherapy in HL is primarily used in young patients with mediastinal and bulky disease. Early relapse-free survival rates are similar to those reported with photon radiation treatment, and no early grade 3 toxicities have been observed. Continued follow-up to assess late effects is critical.

Proton pencil beam scanning for mediastinal lymphoma: the impact of interplay between target motion and beam scanning.

The purpose of this study was to assess the feasibility of proton pencil beam scanning (PBS) for the treatment of mediastinal lymphoma. A group of 7 patients of varying tumor size (100-800 cc) were planned using a PBS anterior field. We investigated 17 fractions of 1.8 Gy(RBE) to deliver 30.6 Gy(RBE) to the internal target volume (ITV). Spots with σ ranging from 4 mm to 8 mm were used for all patients, while larger spots (σ = 6-16 mm) were employed for patients with motion perpendicular to the beam (⩾5 mm), based on initial 4-dimensional computed tomography (4D CT) motion evaluation. We considered volumetric repainting such that the same field would be delivered twice in each fraction. The ratio of extreme inhalation amplitude and regular tidal inhalation amplitude (free-breathing variability) was quantified as an indicator of potential irregular breathing during the scanning. Four-dimensional dose was calculated on the 4D CT scans based on the respiratory trace and beam delivery sequence, implemented by partitioning the spots into separate plans on each 4D CT phase. Four starting phases (end of inhalation, end of exhalation, middle of inhalation and middle of exhalation) were sampled for each painting and 4 energy switching times (0.5 s, 1 s, 3 s and 5 s) were tested, which resulted in 896 dose distributions for the analyzed cohort. Plan robustness was measured for the target and critical structures in terms of the percent difference between 'delivered' dose (4D-evaluated) and planned dose (calculated on average CT). It was found that none of the patients exhibited highly variable or chaotic breathing patterns. For all patients, the ITV D98% was degraded by <2% (standard deviations âˆ¼ 0.1%) when averaged over the whole treatment course. For six out of seven patients, the average degradation of ITV D98% per fraction was within 5% . For one patient with motion perpendicular to the beam (⩾5 mm), the degradation of ITV D98% per fraction was up to 15%, which was mitigated to 2% by employing larger spots and repainting. Deviation of mean lung dose was at most 0.2 Gy(RBE) (less than 1% of prescribed dose, 30.6 Gy(RBE)), while the deviation of heart maximum dose and cord maximum dose could exceed 5% of the prescribed dose. No significant difference in either target coverage or normal tissue dose was observed for different energy switching times compared via two-sided Wilcoxon signed-rank tests (p < 0.05). This feasibility study demonstrates that, for mediastinal lymphoma, the impact of the interplay effect on the PBS plan robustness is minimal when volumetric repainting and/or larger spots are employed.

Dosimetric consequences of pancreatic tumor motion when predetermined treatment margins are employed during intensity-modulated radiation therapy.

PURPOSE: To quantify the dosimetric consequences of pancreatic tumor motion on the pancreatic intensity-modulated radiation therapy (IMRT) plans. METHODS: Dose map of IMRT plans for 5 patients with pancreatic cancer were measured using a 2D diode array placed on a computer-controlled platform to simulate 2D pancreatic tumor motion. Dosimetric analysis was then performed to obtain IMRT quality assurance (QA) passing rates. The convolution method, which used a motion kernel to simulate 2D pancreatic motion, was also applied to the treatment and phantom verification plans for a wide range of magnitudes of motion (0.8-2.0 cm). The resulting motion-convolved verification dose maps (VDMs) were compared with the dynamic measurements to evaluate IMRT QA passing rates as well as the dose-volume histogram, the V95% of the planning target volume (PTV) and V98% of the clinical target volume (CTV). RESULTS: While CTV coverage was maintained when the simulated pancreatic tumor drifted inside the PTV with magnitudes of 1.0 cm and 1.5 cm, the V95% of the PTV was reduced by 10% and 17%, respectively. We also found that the differences between the measurements and the static VDMs increased proportional to the amplitude of motion, while the agreement between the measurements and the motion-convolved VDMs was excellent for any magnitude of motion. CONCLUSIONS: When the 4D technique is not available, predetermined margins must be used carefully to avoid possible under-dose to the target. Additionally, the phantom results show that the kernel convolution method provides an accurate evaluation of the dosimetric impact due to tumor motion and it should be employed in the planning process.

Toxicity data for preoperative concurrent chemoradiotherapy with oxaliplatin and continuous infusion 5-fluorouracil for locally advanced esophageal cancer.

The purpose of this retrospective analysis was to characterize the feasibility and tolerability of oxaliplatin/5-fluorouracil (5-FU) given concurrently with radiotherapy for patients with locally advanced esophageal cancer. Between July 2005 and March 2009, 15 patients with clinical stage T3/T4 and/or N1/M1a lower esophageal or gastroesophageal junction adenocarcinoma were treated with preoperative chemoradiotherapy using oxaliplatin every 2 weeks and continuous infusion 5-FU. The main treatment-related toxicities were oral mucositis and dysphagia. During the first 2 weeks of treatment, 20% of patients presented with grade 1-2 oral mucositis, and one patient developed grade 1 dysphagia. In weeks 3-4, 53% of the patients experienced grade 1-2 mucositis, and 40% experienced grade 1-2 dysphagia. One patient only experienced grade 3 mucositis in week 4. Three patients (20%) had grade 3-4 dysphagia in weeks 3-4 and were continued on intravenous fluids and pain medications. During the last 2 weeks of chemoradiotherapy, 53% of patients reported grade 1-2 oral mucositis, mostly grade 1 and 73% of patients experienced grade 1-2 dysphagia and 26% patients experienced grade 3-4 dysphagia. Other toxicities included fatigue, nausea, neuropathy, and diarrhea. Only one patient experienced > 10% weight loss. The whole group was treated with aggressive supportive care during radiotherapy. Five (33%) patients achieved a pathological complete response. No patients developed locoregional failure. Sixty percent of the patients developed distant metastases and the 2-year disease-free survival was 53%. The median survival was 3.2 years with the 2-year overall survival of 73%. Preoperative oxaliplatin/5-FU-based chemoradiotherapy for locally advanced esophageal cancer is feasible, but associated with substantial gastrointestinal toxicity. A careful attention to nutrition and hydration throughout the course of therapy is required.

TBI during BM and SCT: review of the past, discussion of the present and consideration of future directions.

TBI has been used widely in the setting of BMT over the past 3 decades. Early research demonstrated feasibility and efficacy in the myeloablative setting, in preparation first for allogenic BMT and later for autologous stem cell rescue. As experience with TBI increased, its dual roles of myeloablation and immunosuppression came to be recognized. Toxicity associated with myeloablative TBI remains significant, and this treatment is generally reserved for younger patients with excellent performance status. Reduced intensity conditioning regimens may be useful to provide immunosuppression for patients who are not candidates for myeloablative treatment. Efforts to reduce toxicity through protection of normal tissue using methods of normal tissue blocking and use of TLI, rather than TBI, continue. In the future, modalities such as helical tomotherapy, proton radiotherapy and radioimmunotherapy, may have roles in delivery of radiation to the BM and lymphoid structures with reduced normal tissue toxicity. With further investigation, these efforts may expand the therapeutic ratio associated with TBI, allowing safer delivery to a broader range of patients.

Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning.

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.

Cyclooxygenase-2, malondialdehyde and pyrimidopurinone adducts of deoxyguanosine in human colon cells.

Cyclooxygenases (COX) catalyse the oxygenation of arachidonic acid to prostaglandin (PG) endoperoxides. Activity of one of the COX isoforms, COX-2, results in production of prostaglandin E(2) (PGE(2)) via the endoperoxide PGH(2). COX-2 has been implicated in the pathogenesis of colorectal cancer. Malondialdehyde (MDA) is a mutagen produced by spontaneous and enzymatic breakdown of PGH(2). MDA reacts with DNA to form adducts, predominantly the pyrimidopurinone adduct of deoxyguanosine (M(1)G). Here the hypothesis was tested that COX-2 activity in human colon cells results in formation of MDA and generation of M(1)G adducts. M(1)G was detected in basal cultures of human non-malignant colon epithelial (HCEC) and malignant SW48, SW480, HT29 and HCA-7 colon cells, at levels from 77 to 148 adducts/10(8) nucleotides. Only HCA-7 and HT29 cells expressed COX-2 protein. Levels of M(1)G correlated significantly (r = 0.98, P < 0.001) with those of intracellular MDA determined colorimetrically in the four malignant cell types, but neither parameter correlated with expression of COX-2 or PG biosynthesis. Induction of COX-2 expression by phorbol 12-myristate 13-acetate in HCEC cells increased PGE(2) production 20-fold and MDA concentration 3-fold. Selective inhibition of COX-2 activity in HCA-7 cells by NS-398 significantly inhibited PGE(2) production, but altered neither MDA nor M(1)G levels. Malondialdehyde treatment of HCEC cells resulted in a doubling of M(1)G levels. These results show for the first time in human colon cells that COX-2 activity is associated with formation of the endogenous mutagen, MDA. Moreover, they demonstrate the correlation between MDA concentration and M(1)G adduct levels in malignant cells.

Endogenous DNA damage and mutation.

In humans, approximately 10(7) cells divide per second. Estimates suggest that spontaneous mutations arise in about a third of those cells. These mutations arise as mistakes in DNA replication and when DNA polymerases copy damaged templates. The latter result from chemical hydrolysis of nucleoside bases or by reaction of DNA with electrophiles or reactive free radicals generated during metabolism (endogenous DNA damaging agents). This article highlights recent discoveries and emerging opportunities in the study of endogenous DNA damage and mutation.

Measurement of a malondialdehyde-DNA adduct.

Determining the levels of various DNA adducts has become an essential tool in understanding the toxicology of carcinogens. Direct measurement of DNA adduct levels, the true biologically effective dose of a mutagen, can be correlated with biological outcomes or used to probe mechanisms of adduct formation. Each adduct to be measured requires a specific assay. Malondialdehyde is a carcinogenic and mutagenic electrophile that is endogenously produced during peroxidation of polyunsaturated fatty acids. Its reaction with deoxyguanosine produces a fluorescent exocyclic pyrimidopurinone that can be detected by gas chromatographic/negative chemical ionization-electron capture mass spectroscopy. Methods for preparing an immunoaffinity gel and for HPLC quantification of nucleosides are also included.

Reactivity and mutagenicity of endogenous DNA oxopropenylating agents: base propenals, malondialdehyde, and N(epsilon)-oxopropenyllysine.

Malondialdehyde (MDA), a mutagenic product of lipid peroxidation, reacts with DNA to form the premutagenic lesion, pyrimido[1, 2-a]purin-10(3H)-one (M(1)G). M(1)G is present in normal human tissues, but the contribution of other endogenously produced MDA analogues is poorly understood. Oxidation of the DNA backbone can cause strand breaks and release base propenals, and MDA condensation with proteins yields N(epsilon)-oxopropenyllysine. Here we compare the M(1)G-forming ability and Salmonella typhimurium mutagenicity of MDA with adenine, thymine, and cytosine propenals and N(alpha)-acetyl-N(epsilon)-oxopropenyllysine methyl ester. Base propenals are 30-150 times more potent than MDA in M(1)G formation and are 30-60 times more mutagenic than MDA. In addition, the Fe-bleomycin complex, which generates base propenals, induced M(1)G, but gamma-radiation, which generates mostly MDA, did not. M(1)G formation by MDA and base propenals was concentration-dependent, time-dependent, and enhanced by acidic conditions. N(alpha)-Acetyl-N(epsilon)-oxopropenyllysine methyl ester was less reactive and less mutagenic than MDA. These differences in potency are consistent with differences in leaving group ability. This work supports a role for other MDA analogues, especially base propenals, in the formation of endogenous M(1)G adducts.

Xenobiotic-metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde.

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 >> 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.

A sensitive immunoslot-blot assay for detection of malondialdehyde-deoxyguanosine in human DNA.

As part of a large programme on food risk assessment, we have become Interested in the endogenous production of genotoxic agents from dietary precursors. Malondialdehyde (MDA), a product of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in bacterial and mammalian systems. MDA reacts with DNA, and the major adduct (M1-dG) has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to large numbers of individuals or a large variety of samples. Often, only a few micrograms of DNA from human tissues are available for analysis, and a very sensitive assay is needed to detect background levels of M1-dG in very small amounts of DNA. In this paper, we describe the development of an immunoslot-blot (ISB) assay for the measurement of M1-dG in 1 microgram of DNA. The limit of detection of the assay is about 5 adducts per 10(8) bases. The advantages of ISB over other assays for DNA adduct detection, such as the possibility of analysing 1 microgram DNA per sample and the fact that it is less time-consuming and laborious, mean that it can be more easily used for routine analysis of large numbers of samples in biomonitoring. A series of human samples was analysed, and levels of 0.3-6.43 M1-dG per 10(7) normal bases were detected in 42 gastric biopsy samples and 0.7-16.65 M1-dG per 10(7) normal bases in 28 samples of leukocyte DNA. In an initial study in five human volunteers on standardized diets, the levels of M1-dG in leukocyte DNA changed in relation to meat, vegetable and tea intake.

Determination of malondialdehyde-induced DNA damage in human tissues using an immunoslot blot assay.

Malondialdehyde (MDA) is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic and the major adduct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M1-dG), has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to a large number of individuals or variety of samples. Often, only a few microg of DNA from human tissues are available for analysis and a very sensitive assay is needed in order to detect background levels of M1-dG in very small amounts of DNA. In this paper, the development of an immunoslot blot (ISB) assay for the measurement of MI-dG in 1 microg of DNA is described. The limit of detection of the assay is 2.5 adducts per 10(8) bases. A series of human samples were analysed and levels of 5.6-9.5 (n = 8) and 3.1-64.3 (n = 42) of M1-dG per 10(8) normal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those obtained using an HPLC/32P-post-labelling (HPLC/PPL) method previously developed and indicated a high correlation between M1-dG levels measured by the two assays. The advantages of ISB over other assays including HPLC/PPL, such as the possibility of analysing 1 microg DNA/sample and the fact that it is less time-consuming and laborious, means that it can be more easily used for routine analysis of a large number of samples in biomonitoring studies.

Comparison of DNA adduct levels associated with oxidative stress in human pancreas.

DNA adducts associated with oxidative stress are believed to involve the formation of endogenous reactive species generated by oxidative damage and lipid peroxidation. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been carried out. In this study, we isolated DNA from the pancreas of 15 smokers and 15 non-smokers, and measured the levels of 1,N6-etheno(2'-deoxy)guanosine (edA), 3, N4-etheno(2'-deoxy)cytidine (edC), 8-oxo-2'-deoxyguanosine (8-oxo-dG), and pyrimido[1,2-alpha]purin-10(3H)-one (m1G). Using the same DNA, the glutathione S-transferase (GST) M1, GSTT1, and NAD(P)H quinone reductase-1 (NQO1) genotypes were determined in order to assess the role of their gene products in modulating adduct levels through their involvement in detoxification of lipid peroxidation products and redox cycling, respectively. The highest adduct levels observed were for m1G, followed by 8-oxo-dG, edA, and edC, but there were no differences in adduct levels between smokers and non-smokers and no correlation with the age, sex or body mass index of the subject. Moreover, there was no correlation in adduct levels between edA and eC, or between edA or edC and m1G or 8-oxo-dG. However, there was a significant correlation (r=0.76; p<0.01) between the levels of 8-oxo-dG and m1G in human pancreas DNA. Neither GSTM1 nor NQO1 genotypes were associated with differences in any of the adduct levels. Although the sample set was limited, the data suggest that endogenous DNA adduct formation in human pancreas is not clearly derived from cigarette smoking or from (NQO1)-mediated redox cycling. Further, it appears that neither GSTM1 nor GSTT1 appreciably protects against endogenous adduct formation. Together with the lack of correlation between m1G and edA or edC, these data indicate that the malondialdehyde derived from lipid peroxidation may not contribute significantly to m1G adduct formation. On the other hand, the apparent correlation between m1G and 8-oxo-dG and their comparable high levels are consistent with the hypothesis that m1G is formed primarily by reaction of DNA with a base propenal, which, like 8-oxo-dG, is thought to be derived from hydroxyl radical attack on the DNA.

Indirect mutagenesis by oxidative DNA damage: formation of the pyrimidopurinone adduct of deoxyguanosine by base propenal.

Oxidation of endogenous macromolecules can generate electrophiles capable of forming mutagenic adducts in DNA. The lipid peroxidation product malondialdehyde, for example, reacts with DNA to form M1G, the mutagenic pyrimidopurinone adduct of deoxyguanosine. In addition to free radical attack of lipids, DNA is also continuously subjected to oxidative damage. Among the products of oxidative DNA damage are base propenals. We hypothesized that these structural analogs of malondialdehyde would react with DNA to form M1G. Consistent with this hypothesis, we detected a dose-dependent increase in M1G in DNA treated with calicheamicin and bleomycin, oxidizing agents known to produce base propenal. The hypothesis was proven when we determined that 9-(3-oxoprop-1-enyl)adenine gives rise to the M1G adduct with greater efficiency than malondialdehyde itself. The reactivity of base propenals to form M1G and their presence in the target DNA suggest that base propenals derived from oxidative DNA damage may contribute to the mutagenic burden of a cell.