Mark E. Bernard

Nitric Oxide Is a Key Component in Inflammation-Accelerated Tumorigenesis

Nitric oxide (NO(*)), an important signaling molecule and a component of inflammatory response, is involved in tumorigenesis. However, the quantity of NO(*) and the cellular microenvironment influences the role of NO(*) in tumor development. We used a genetic strategy to test the hypothesis that an inflammatory microenvironment with an enhanced level of NO(*) accelerates spontaneous tumor development. C. parvum-induced inflammation and increased NO(*) synthase-2 (NOS2) expression coincided with accelerated spontaneous tumor development, mostly lymphomas, in p53-/-NOS2+/+ C57BL6 mice when compared with the controls (P = 0.001). However, p53-/-NOS2-/- mice did not show any difference in tumor latency between C. parvum-treated and control groups. In C. parvum-treated p53-/-NOS2+/+ mice, tumor development was preceded by a higher expression of NOS2 and phosphorylated Akt-Ser(473) (pAkt-Ser473) in spleen, increased cell proliferation measured by Ki-67 IHC in spleen and thymus, and a lower apoptotic index and CD95-L expression in spleen and thymus. C. parvum-treated p53-/-NOS2+/+ mice showed an increase in the number of Foxp3(+) T-reg cells, dendritic cells (DC), as well as increased CD80(+), CD86(+), CD40(+), and CD83(+) on DC in the spleen. Regulatory T-cells (T-reg) and the maturation of DC may modulate tumorigenesis. An increase in the FoxP3(+)T-reg cells in C. parvum-treated p53-/-NOS2+/+ mice indicates a role of NO(*) in the regulation of T-reg cells that may contribute to a protumor shift of the immune environment favoring an accelerated tumor development. These data provide genetic and mechanistic evidence that an inflammatory microenvironment and an increased level of NO(*) can accelerate tumor development.

Quantitative Detection of p53 Mutations in Plasma DNA from Tobacco Smokers

In lung tumors, the p53 tumor suppressor gene is commonly mutated with a characteristic mutation spectrum. The amount of and alterations in plasma DNA, such as mutations in p53, were associated with several cancers. Few studies used quantitative methods of high sensitivity. Previously, we observed p53 mutations in the noncancerous tissue that differed from those in lung tumors using the highly sensitive p53 mutation load assay. Based on our observation of an increased p53 mutation load in nontumorous lung tissue in smokers, we hypothesized that plasma DNA may contain mutant p53 indicative of tobacco smoke exposure and will be an effective biomarker of lung cancer or smoking exposure. We modified the p53 mutation load assay to detect mutations at p53 codons 248 and 249, common mutations in lung cancer, in plasma DNA samples with a sensitivity of 1:5,000. The assay was applied to a set of lung cancer cases (n = 39), hospital controls (n = 21), and population controls (n = 20) from a larger study. Controls were selected to consist of equal numbers of both ever and never smokers. The p53 mutation load (mutated p53 copies per total number of p53 copies) was associated with smoking (P = 0.06), but not with lung cancer (P = 0.59). Most of the individuals with p53 mutations observed in plasma DNA were ever smokers and the p53 mutation load was higher in those who smoked for longer durations (P = 0.04). In summary, we were able to detect p53 mutations in plasma DNA from healthy individuals and our data suggest that p53 mutations in plasma DNA may be a marker of carcinogen exposure from tobacco smoke.

Impact of adjuvant chemotherapy with radiation for node-positive vulvar cancer: A National Cancer Data Base (NCDB) analysis ☆

BACKGROUND For node-positive vulvar cancer, adjuvant radiotherapy has an established benefit, whereas the impact of chemotherapy is unknown. A National Cancer Data Base (NCDB) analysis was conducted to determine patterns of care and evaluate the survival impact of adjuvant chemotherapy. METHODS The NCDB was queried for vulvar cancer patients diagnosed from 1998-2011 who underwent extirpative surgery with confirmed inguinal nodal involvement treated with adjuvant radiotherapy. Patients with inadequate follow-up or non-squamous histologies were excluded. Chi-square test, logistic regression analysis, log-rank test and multivariable Cox proportional regression modeling with adjustment using propensity score with inverse probability of treatment weights (IPTW) were conducted to establish factors associated with utilization and survival. RESULTS A total of 1797 patients were identified: 26.3% received adjuvant chemotherapy and 76.6% had 1-3 involved lymph nodes. Adoption of adjuvant chemotherapy significantly increased over time, from 10.8% in 1998 to 41.0% in 2006 (p<0.001). Lower utilization was seen in older patients, Northeast or Southern facilities, and patients with more extensive nodal dissection, whereas greater number of involved nodes, stage IVA disease and positive surgical margins led to a higher probability of receiving chemotherapy. Unadjusted median survival without and with adjuvant chemotherapy was 29.7months and 44.0months (p=0.001). On IPTW-adjusted Cox proportional regression modeling, delivery of adjuvant chemotherapy resulted in a 38% reduction in the risk of death (HR 0.62, 95% CI 0.48-0.79, p<0.001). CONCLUSION In a large population-based analysis, adjuvant chemotherapy resulted in a significant reduction in mortality risk for node-positive vulvar cancer patients who received adjuvant radiotherapy.

The autophagy-inducing drug carbamazepine is a radiation protector and mitigator

Abstract Purpose: To determine whether Carbamazepine (CBZ) is a radiation protector and/or mitigator. Materials and methods: Murine hematopoietic progenitor 32D cl 3 cells were incubated in 1, 10, or 100 μM CBZ 1 h before or immediately after 0–8 Gy irradiation and assayed for clonogenic survival. Autophagy was assayed by immunoblot for microtubule-associated protein light chain 3 (LC3). In vivo radioprotection and mitigation were determined with C57BL/6NTac mice. Results: CBZ treatment at 1, 10 or 100 μM for 1 h prior to irradiation increased radioresistance (the dose for 37% survival or D0) from control 1.5 ± 0.1 Gy to 2.1 ± 0.2 Gy (P = 0.012), 2.3 ± 0.1 Gy (P = 0.010), and 3.6 ± 0.7 Gy (P = 0.003), respectively; after irradiation increased the extrapolation number (ñ) from 1.5 ± 0.3 to 10.1 ± 4.2 (P = 0.011), 5.5 ± 1.7 (P = 0.019), and 3.6 ± 0.8 (P = 0.014), respectively, and increased autophagy. CBZ treated mice 10 min or 24 h before or 10 min or 12 h after 9.25 Gy total body irradiation (TBI) showed increased survival (P = 0.012, 0.011, 0.0002, and 0.017, respectively). Conclusion: CBZ may be a useful radiation protector and mitigator.

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines.

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG–/– (PD326) and FancD2–/– (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2–/– cells were more radiosensitive than the transgene restored subclonal cell line (ñ = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG–/– cells were radioresistant relative to the transgene-restored subclonal cell line (ñ = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2–/– cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2–/– cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.

Stereotactic body radiotherapy for the treatment of presacral recurrences from rectal cancers

PURPOSE Management of recurrent presacral rectal cancer is often not amenable to curative surgery. The goal of this study is to evaluate the safety and efficacy of cyberknife stereotactic body radiotherapy (SBRT) in the management of presacral recurrences. MATERIALS AND METHODS Between April 2003 and October 2008, 14 patients with presacral tumors from rectal adenocarcinoma were SBRT treated. Eleven patients were treated with 36 Gy in 3 fractions and 3 patients were treated with single fraction of 12, 16 or 18 Gy. Tumor response was assessed using response evaluation and criteria in solid tumor (RECIST) criteria. Toxicities were assessed with common terminology criteria adverse events v 3.0. Pain control was assessed. RESULTS One patient (6.7%) received SBRT as boost therapy. All patients had prior radiotherapy [median 50.4 Gy (20 - 81 Gy)]. Median tumor volume was 52.5 cc (19 - 110 cc). At initial follow-up of a median 4.9 months (1 - 16.3 months), treatment responses were complete response (n=3) and stable disease (n=8). With a median follow-up of 16.5 months (6 - 69 months), the one- and two-year LC rates were 90.9 and 68.2%, respectively, and the one- and two-year OS rates were 90 and 78.8%, respectively. No factors were significantly predictive of LC and OS. There were no grade 3 or 4 toxicities. Fifty percent (n=7) of our patients experienced pain with recurrence before treatment and 4 (57.1%) of them reported no pain after completion of their SBRT. CONCLUSIONS Stereotactic body radiotherapy for presacral recurrence of rectal adenocarcinoma is an efficacious and well-tolerated treatment modality which allows for palliation of pain.

Linear accelerator based stereotactic radiosurgery for melanoma brain metastases.

PURPOSE Melanoma is one of the most common malignancies to metastasize to the brain. Many patients with this disease will succumb to central nervous system (CNS) disease, highlighting the importance of effective local treatment of brain metastases for both palliation and survival of the disease. Our objective was to evaluate the outcomes associated with stereotactic radiosurgery (SRS) in the treatment of melanoma brain metastases. MATERIALS AND METHODS We retrospectively reviewed 54 patients with a total of 103 tumors treated with SRS. Twenty patients had prior surgical resection and nine patients underwent prior whole brain radiation therapy (WBRT). 71% of patients had active extracranial disease at the time of SRS. Median number of tumors treated with SRS was 1(range: 1-6) with median radiosurgery tumor volume 2.1 cm 3 (range: 0.05-59.7 cm 3 ). The median dose delivered to the 80% isodose line was 24 Gy in a single fraction. RESULTS The median follow-up from SRS was five months (range:1-30 months). Sixty-five percent of patients had a follow-up MRI available for review. Actuarial local control at six months and 12 months was 87 and 68%, respectively. Eighty-one percent of patients developed new distant brain metastases at a median time of two months. The six-month and 12-month actuarial overall survival rates were 50 and 25%, respectively. The only significant predictor of overall survival was surgical resection prior to SRS. Post-SRS bleeding occurred in 18% of patients and at a median interval of 1.5 months. There was only one episode of radiation necrosis with no other treatment-related toxicity. CONCLUSION SRS for brain metastases from melanoma is safe and achieves acceptable local control.

Salvage stereotactic radiosurgery for recurrent glioblastoma multiforme with prior radiation therapy

BACKGROUND Glioblastoma multiforme (GBM) carries a poor prognosis with high recurrence rates. Salvage stereotactic radiosurgery (SRS) may be an effective treatment option. METHODS We retrospectively reviewed 34 patients (41 lesions) treated with salvage SRS for recurrent GBM between 2004 and 2012. Initial surgical treatments were gross total resection (58%), subtotal resection (STR) (24%), and biopsy (18%). All patients were treated with prior radiation therapy. Recurrent disease was treated with salvage SRS with a median dose and fractions of 23.4 Gy (range, 12-30) and 3 (range, 1-3), respectively. Cox proportional hazards regression was conducted to establish predictive factors (P ≤ 0.05) Results: Median follow-up from salvage SRS was 10.8 months (interquartile range [IQR], 7.0-15.6). The median time from initial radiation therapy to salvage SRS was 13.7 months (IQR, 2.9-25.0). The 6- and 12-month overall survival from salvage SRS were 84.9% and 42.5%, respectively. On univariate analysis, STR was associated with inferior survival from salvage SRS (P ≤ 0.05). The 6- and 12-month local control (LC) estimates were 63.1% and 16.4%, respectively. On univariate analysis, higher biological effective dose and prior temozolomide were associated with superior LC. Concerning toxicity, there were 4 (12%) grade 2 and 1 (3%) grade 3 adverse events within this patient series. No grade 4 or grade 5 toxicities were observed. CONCLUSION Our outcomes suggest that SRS is a feasible treatment option with acceptable salvage survival rates, given the poor prognosis of this disease.

One- vs. Three-Fraction Pancreatic Stereotactic Body Radiation Therapy for Pancreatic Carcinoma: Single Institution Retrospective Review

Background/introduction Early reports of stereotactic body radiation therapy (SBRT) for pancreatic ductal adenocarcinoma (PDAC) used single fraction, but eventually shifted to multifraction regimens. We conducted a single institution review of our patients treated with single- or multifraction SBRT to determine whether any outcome differences existed. Methods and materials Patients treated with SBRT in any setting for PDAC at our facility were included, from 2004 to 2014. Overall survival (OS), local control (LC), regional control (RC), distant metastasis (DM), and late grade 3 or greater radiation toxicities from the time of SBRT were calculated using Kaplan–Meier estimation to either the date of last follow-up/death or local/regional/distant failure. Results We identified 289 patients (291 lesions) with pathologically confirmed PDAC. Median age was 69 (range, 33–90) years. Median gross tumor volume was 12.3 (8.6–21.3) cm3 and planning target volume 17.9 (12–27) cm3. Single fraction was used in 90 (30.9%) and multifraction in 201 (69.1%) lesions. At a median follow-up of 17.3 months (IQR 10.1–29.3 months), the median survival for the entire cohort 17.8 months with a 2-year OS of 35.3%. Univariate analysis showed multifraction schemes to have a higher 2-year OS 30.5% vs. 37.5% (p = 0.019), it did not hold significance on MVA. Multifractionation schemes were found to have a higher LC on MVA (HR = 0.53, 95% CI, 0.33–0.85, p = 0.009). At 2 years, late grade 3+ toxicity was 2.5%. Post-SBRT CA19-9 was found on MVA to be a prognostic factor for OS (HR = 1.01, 95% CI, 1.01–1.01, p = 0.009), RC (HR = 1.01, 95% CI 1.01–1.01, p = 0.02), and DM (HR = 1.01, 95% CI, 1.01–1.01, p = 0.001). Conclusion Our single institution retrospective review is the largest to date comparing single and multifraction SBRT and the first to show multifraction regimen SBRT to have a higher LC than single fractionation. Additionally, we show low rates of severe late toxicity with SBRT.

Can Radiosensitivity Associated with Defects in DNA Repair be Overcome by Mitochondrial-Targeted Antioxidant Radioprotectors

Radiation oncologists have observed variation in normal tissue responses between patients in many instances with no apparent explanation. The association of clinical tissue radiosensitivity with specific genetic repair defects (Wegner’s syndrome, Ataxia telangiectasia, Bloom’s syndrome, and Fanconi anemia) has been well established, but there are unexplained differences between patients in the general population with respect to the intensity and rapidity of appearance of normal tissue toxicity including radiation dermatitis, oral cavity mucositis, esophagitis, as well as differences in response of normal tissues to standard analgesic or other palliative measures. Strategies for the use of clinical radioprotectors have included modalities designed to either prevent and/or palliate the consequences of radiosensitivity. Most prominently, modification of total dose, fraction size, or total time of treatment delivery has been necessary in many patients, but such modifications may reduce the likelihood of local control and/or radiocurability. As a model system in which to study potential radioprotection by mitochondrial-targeted antioxidant small molecules, we have studied cell lines and tissues from Fanconi anemia (Fancd2−/−) mice of two background strains (C57BL/6NHsd and FVB/N). Both were shown to be radiosensitive with respect to clonogenic survival curves of bone marrow stromal cells in culture and severity of oral cavity mucositis during single fraction or fractionated radiotherapy. Oral administration of the antioxidant GS-nitroxide, JP4-039, provided significant radioprotection, and also ameliorated distant bone marrow suppression (abscopal effect of irradiation) in Fancd2−/− mice. These data suggest that radiation protection by targeting the mitochondria may be of therapeutic benefit even in the setting of defects in the DNA repair process for irradiation-induced DNA double strand breaks.