Ronny Kalash

Prospective evaluation of patient-reported quality-of-life outcomes following SBRT ± cetuximab for locally-recurrent, previously-irradiated head and neck cancer

PURPOSE Stereotactic body radiotherapy (SBRT) has emerged as a promising salvage strategy for unresectable, previously-irradiated recurrent squamous cell carcinomas of the head and neck (rSCCHN). Here-in, we report the first prospective evaluation of patient-reported quality-of-life (PR-QoL) following re-irradiation with SBRT±cetuximab for rSCCHN. MATERIALS AND METHODS From November 2004 to May 2011, 150 patients with unresectable, rSCCHN in a previously-irradiated field receiving >40 Gy were treated with SBRT to 40-50 Gy in 5 fractions ± concurrent cetuximab. PR-QoL was prospectively acquired using the University of Washington Quality-of-Life Revised (UW-QoL-R). RESULTS Overall PR-QoL, health-related PR-QoL, and select domains commonly affected by re-irradiation progressively increase following an initial 1-month decline with statistically significant improvements noted in swallowing (p=0.025), speech (p=0.017), saliva (p=0.041), activity (p=0.032) and recreation (p=0.039). CONCLUSIONS Especially for patients surviving >1-year, improved tumor control associated with SBRT re-irradiation may ameliorate decreased PR-QoL resulting from rSCCHN. These improvements in PR-QoL transcend all measured domains in a validated PR-QoL assessment tool independent of age, use of cetuximab, tumor volume, and interval since prior irradiation.

Examining tumor control and toxicity after stereotactic body radiotherapy in locally recurrent previously irradiated head and neck cancers: Implications of treatment duration and tumor volume

Stereotactic body radiotherapy (SBRT) has been studied in locally recurrent previously‐irradiated head and neck cancers; however, the optimum fractionation and patient selection continues to be defined.

Radiologic Differences between Bone Marrow Stromal and Hematopoietic Progenitor Cell Lines from Fanconi Anemia (Fancd2–/–) Mice

FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2–/– mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2–/– mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2–/– mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2+/+ stromal cells (Fancd2–/– D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2+/+ D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2–/– IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2+/+ (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2–/– marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2–/– stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2+/+ cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2–/– IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2–/– stromal cells, hematopoietic progenitor cells showed reduced G2/M cell cycle arrest. The absence of the mouse Fancd2 gene product confers radiosensitivity to bone marrow stromal but not hematopoietic progenitor cells.

Conditional Radioresistance of tet-Inducible Manganese Superoxide Dismutase Bone Marrow Stromal Cell Lines

Mitochondrial targeted manganese superoxide dismutase is a major antioxidant enzyme, the levels of which modulate the response of cells, tissues and organs to ionizing irradiation. We developed a Tet-regulated MnSOD mouse (MnSODtet) to examine the detailed relationship between cellular MnSOD concentration and radioresistance and carried out in vitro studies using bone marrow culture derived stromal cell lines (mesenchymal stem cells). Homozygous MnSODtet/tet cells had low levels of MnSOD, reduced viability and proliferation, increased radiosensitivity, elevated overall antioxidant stores, and defects in cell proliferation and DNA strand-break repair. Doxycycline (doxy) treatment of MnSODtet/tet cells increased MnSOD levels and radioresistance from ñ of 2.79 ± 1.04 to 8.69 ± 1.09 (P = 0.0060) and normalized other biologic parameters. In contrast, MnSODtet/tet cells showed minimal difference in baseline and radiation induced mRNA and protein levels of TGF-β, Nrf2 and NF-κB and radiation induced cell cycle arrest was not dependent upon MnSOD level. These novel MnSODtet/tet mouse derived cells should be valuable for elucidating several parameters of the oxidative stress response to ionizing radiation.

Amelioration of Radiation-Induced Oral Cavity Mucositis and Distant Bone Marrow Suppression in Fanconi Anemia Fancd2–/– (FVB/N) Mice by Intraoral GS-Nitroxide JP4-039

The altered DNA damage response pathway in patients with Fanconi anemia (FA) may increase the toxicity of clinical radiotherapy. We quantitated oral cavity mucositis in irradiated Fanconi anemia Fancd2–/– mice, comparing this to Fancd2+/– and Fancd2+/+ mice, and we measured distant bone marrow suppression and quantitated the effect of the intraoral radioprotector GS-nitroxide, JP4-039 in F15 emulsion. We found that FA mice were more susceptible to radiation injury and that protection from radiation injury by JP4-039/F15 was observed at all radiation doses. Adult 10–12-week-old mice, of FVB/N background Fancd2–/–, Fancd2+/– and Fancd2+/+ were head and neck irradiated with 24, 26, 28 or 30 Gy (large fraction sizes typical of stereotactic radiosurgery treatments) and subgroups received intraoral JP4-039 (0.4 mg/mouse in 100 μL F15 liposome emulsion) preirradiation. On day 2 or 5 postirradiation, mice were sacrificed, tongue tissue and femur marrow were excised for quantitation of radiation-induced stress response, inflammatory and antioxidant gene transcripts, histopathology and assay for femur marrow colony-forming hematopoietic progenitor cells. Fancd2–/– mice had a significantly higher percentage of oral mucosal ulceration at day 5 after 26 Gy irradiation (59.4 ± 8.2%) compared to control Fancd2+/+ mice (21.7 ± 2.9%, P = 0.0063). After 24 Gy irradiation, Fancd2–/– mice had a higher oral cavity percentage of tongue ulceration compared to Fancd2+/+ mice irradiated with higher doses of 26 Gy (P = 0.0123). Baseline and postirradiation oral cavity gene transcripts were altered in Fancd2–/– mice compared to Fancd2+/+ controls. Fancd2–/– mice had decreased baseline femur marrow CFU-GM, BFUe and CFU-GEMM, which further decreased after 24 or 26 Gy head and neck irradiation. These changes were not seen in head- and neck-irradiated Fancd2+/+ mice. In radiosensitive Fancd2–/– mice, biomarkers of both local oral cavity and distant marrow radiation toxicity were ameliorated by intraoral JP4-039/F15. We propose that Fancd2–/– mice are a valuable radiosensitive animal model system, which can be used to evaluate potential radioprotective agents.

Intraoral Mitochondrial-Targeted GS-Nitroxide, JP4-039, Radioprotects Normal Tissue in Tumor-Bearing Radiosensitive Fancd2–/– (C57BL/6) Mice

We evaluated normal tissue specific radioprotection of the oral cavity in radiosensitive Fanconi Anemia (FA) Fancd2–/– mice with orally established tumors using mitochondrial-targeted GS-nitroxide (JP4-039). Adult (10–12 weeks old) Fancd2+/+, Fancd2+/– and Fancd2–/– mice (C57BL/6 background) and subgroups with orally established TC-1 epithelial cell tumors received a single fraction of 28 Gy or four daily fractions of 8 Gy to the head and neck. Subgroups received JP4-039 in F15 emulsion (F15/JP4-039; 0.4 mg/mouse), 4-amino-Tempo in F15 emulsion (F15/4-amino-Tempo; 0.2 mg/mouse) or F15 emulsion alone prior to each irradiation. Oral mucosa of Fancd2–/– mice showed baseline elevated RNA transcripts for Sod2, p53, p21 and Rad51 (all P < 0.0012) and suppressed levels of Nfkb and Tgfb, (all P < 0.0020) compared with Fancd2+/+ mice. The oral mucosa in tumor-bearing mice of all genotypes showed decreased levels of p53 and elevated Tgfb and Gadd45a (P ≤ 0.0001 for all three genotypes). Intraoral F15/JP4-039, but not F15/4-amino-Tempo, modulated radiation-induced normal tissue transcript elevation, ameliorated mucosal ulceration and reduced the depletion of antioxidant stores in oral cavity tissue of all genotypes, but did not radioprotect tumors. Mitochondrial targeting makes F15/JP4-039 an effective normal tissue radioprotector for Fancd2–/– mice, as well as wild-type mice.

Amelioration of Radiation-Induced Pulmonary Fibrosis by a Water-Soluble Bifunctional Sulfoxide Radiation Mitigator (MMS350)

A water-soluble ionizing radiation mitigator would have considerable advantages for the management of acute and chronic effects of ionizing radiation. We report that a novel oxetanyl sulfoxide (MMS350) is effective both as a protector and a mitigator of clonal mouse bone marrow stromal cell lines in vitro, and is an effective in vivo mitigator when administered 24 h after 9.5 Gy (LD100/30) total-body irradiation of C57BL/6NHsd mice, significantly improving survival (P = 0.0097). Furthermore, MMS350 (400 μM) added weekly to drinking water after 20 Gy thoracic irradiation significantly decreased: expression of pulmonary inflammatory and profibrotic gene transcripts and proteins; migration into the lungs of bone marrow origin luciferase+/GFP+ (luc+/GFP+) fibroblast progenitors (in both luc+ marrow chimeric and luc+ stromal cell line injected mouse models) and decreased radiation-induced pulmonary fibrosis (P < 0.0001). This nontoxic and orally administered small molecule may be an effective therapeutic in clinical radiotherapy and as a counter measure against the acute and chronic effects of ionizing radiation.

Abstract 3340: Intraoral administration of mitochondrial targeted GS-nitroxide (JP4-039) radioprotects the oral mucosa but not orthotopic tumors in Fancd2-/- mice

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Fanconi Anemia (FA) patients are at risk for head and neck squamous cell carcinomas. Their normal tissue radiosensitivity often presents a challenge to deliver radiotherapy. Locally applied GS-nitroxide JP4-039 radioprotects the oral mucosa of Fancd2-/-(129/Sv) mice from single fraction or fractionated irradiation (Berhane et al, Radiation Research 182:35-49, 2014). We sought to prove that JP4-039 targets the mitochondria of the oral mucosal cells, and protects normal tissue in vivo, but does not prevent radiocontrolability of tumors. Fancd2+/+, Fancd2+/- and Fancd2-/- mice of a different background strain (C57BL/6), received intraoral flurochrome labelled bodipy-JP4-039 in F15 liposomes, were sacrificed 2 hr later and the oral mucosa removed sectioned, and stained with an antibody to mitochondrial protein TOM-20 and examined microscopically for co-localization of bodipy-JP4-039. Mitochondria were isolated from explanted oral mucosal cells and uptake of JP4-039 compared to non-mitochondrial targeted Tempo, measured using EPR. Fancd2+/+, Fancd2+/- and Fancd2-/- mice with orthotopic TC-1 murine squamous cell tumors were treated with JP-4-039/F15, then irradiated. Mitochondrial uptake in normal tissue but not tumors was demonstrated by co-localization of Bodipy-JP4-039 and mitochondrial protein TOM-20. EPR analysis of purified mitochondria from explanted oral cavity cells of Fancd2+/+ and Fancd2-/- mice treated with F15-JP4-039 showed a 15.6 and 19.1 fold increased uptake of nitroxide signal in JP4-039 treated mice, respectively, compared to Tempo uptake in mitochondria . There was no significant difference in tumor control between the JP4-039/F15 treated then irradiated compared to control irradiated mice, in any of the 3 genotypes: Fancd2+/+ group, p = 0.6851; Fancd2+/− group p = 0.7174 and p = 0.7559 in the Fancd2−/− group. JP4-039/F15 administration prior to either single fraction 28 Gy or prior to each fraction of fractionated head and neck irradiation (8 Gy x 4) showed significant normal tissue protection (decreased ulceration) but not tumor radioprotection. Irradiation reduced tumor volume in mice of all genotypes with no detectable effect of JP4-039/F15. Specifically, with wild type Fancd2+/+ mice after 28 Gy, there was no radioprotective effect of JP4-039/F15 on tumor regrowth (p = 0.7520). Tumor bearing Fancd2−/− (p = 0.1843) and Fancd2+/− mice also showed normal tissue but not tumor protection (p = 0.4106 and p = 0.1843, respectively). The data support use of normal mucosal radioprotective JP4-039/F15 during radiotherapy of FA patients with head and neck cancer. Citation Format: Michael W. Epperly, Ashwin Shinde, Hebist Berhane, Byung Han Rhieu, Ronny Kalash, Karen Xu, Darcy Franicola, Xichen Zhang, Tracy Dixon, Donna Shields, Hong Wang, Peter Wipf, Kalindi Parmar, Eva Guinan, Valerian Kagan, Yulia Tyurina, Robert L. Ferris, Song Li, Joel S. Greenberger. Intraoral administration of mitochondrial targeted GS-nitroxide (JP4-039) radioprotects the oral mucosa but not orthotopic tumors in Fancd2-/- mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2015-3340

Stereotactic Ablative Radiotherapy for Unresectable Colorectal Oligometastases

Purpose Patients with oligometastatic colorectal cancer have demonstrated excellent clinical outcomes with surgical resection of hepatic and pulmonary metastases. Stereotactic ablative radiation therapy (SABR) has emerged as an alternative local therapy for nonsurgical candidates. Herein, we report the oncologic and patient-reported quality-of-life (PR-QoL) outcomes for a subset of patients with oligometastatic colorectal cancer who were treated in a prospective phase 2 multicenter clinical trial. Methods and materials Patients with a pathologically proven diagnosis of oligometastatic colorectal cancer were enrolled as part of a prospective study. SABR dose and fractionation schedules were dependent on the lesion location and size. Patient follow-up occurred 6 weeks after completion of SABR and at 3-month intervals for the following 3 years. Patients received the Functional Assessment of Cancer Therapy-General questionnaire at baseline and at each follow-up visit to assess PR-QoL. The total Functional Assessment of Cancer Therapy-General questionnaire scores were compared with those from baseline using the Wilcoxon signed rank test. Overall survival, local progression-free survival (PFS), and distant PFS were calculated using the Kaplan-Meier estimation to the date of the last follow-up visit/death or local/distant failure. Results A total of 31 patients with oligometastatic colorectal cancer with 1 (71.0%), 2 (16.1%), 3 (3.2%), 4 (3.2%), or 5 (6.5%) metastatic lesions were identified. After a median follow-up time of 50.1 months, the median OS from the time of completion of the SABR was 53.9 months (95% confidence interval, 23.2-84.6), and the 5-year OS, local PFS, and distant PFS were 45%, 83%, and 27%, respectively. Acute grade 2+ toxicity was 9.7% (pain, nausea, fatigue) and late grade 3+ toxicity (small bowel obstruction) was 3.2% with no significant change in PR-QoL in the year after SABR. Conclusions This subset analysis of a prospective phase 2 study demonstrates that SABR is a safe and effective treatment option for patients with unresectable oligometastic colorectal cancer. In addition, SABR of oligometastatic disease preserves PR-QoL.

Exceptional Eight-year Response to Stereotactic Radiosurgery Monotherapy for Multiple Brain Metastases

Breast cancer represents the second leading cause of brain metastases in women. Once diagnosed, brain metastases have been associated with a rapidly progressive and universally poor prognosis. Breast cancer patients, particularly those with advantageous disease characteristics, may achieve extended survival. This extended life expectancy highlights the importance of effective intracranial treatments that minimize treatment-related late toxicity. Whole brain radiation therapy (WBRT) remains a standard of care palliative option; however, concerns remain regarding the late neurocognitive effects. Stereotactic radiosurgery (SRS) provides dose-escalated radiation therapy over a shortened course, maintaining equivalent survival and minimizing normal brain tissue exposure. Herein, we present a breast cancer patient who demonstrated an exceptional response and remained functionally independent following 12 SRS courses targeting 14 unique brain metastases over eight years. The case illustrates the efficacy of SRS alone, as well as the comparable utility of multiple SRS treatment techniques (Gamma Knife (AB Elekta, Stockholm, Sweden), CyberKnife (Accuray, Sunnyvale, California), and TrueBeam (Varian Medical Systems, Palo Alto, California)).