Purpose: Stereotactic body radiation therapy (SBRT) has been established as a safe and effective treatment for prostate cancer. SBRT requires high accuracy to reduce treatment margins. Metal hip prostheses create artifacts that distort pelvic imaging and potentially decrease the accuracy of target/organ at risk (OAR) identification and radiation dose calculations. Data on the safety and efficacy of SBRT after hip replacement is limited. This single -institution study sought to evaluate the safety and local control following SBRT for prostate cancer in men with hip replacements. Methods: 23 patients treated with localized prostate cancer and a history of pre-treatment hip replacement, treated with SBRT from 2007 to 2017 at MedStar Georgetown University Hospital were included in this retrospective analysis. Treatment was administered with the CyberKnife (R) (Accuray Incorporated, Sunnyvale, CA) at doses of 35 Gy or 36.25 Gy in 5 fractions. The targets and OARs were identified and contoured by a single experienced Radiation Oncologist (SPC). The adequacy of the CT and T2W MRI images for treatment planning was assessed with a three-point scale (good, adequate, or suboptimal). During treatment planning, care was taken to avoid treatment beams that directly traversed the hip prosthesis. Toxicities were recorded and scored using the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v.4.0). Local recurrence was confirmed by magnetic resonance imaging and/or prostate biopsy. Results: The median follow-up was seven years. The patients were elderly (median age = 71 years) with a high rate of comorbidities (Charlson Comorbidity Index > 2 in 25%). Four patients had bilateral hip replacements. The majority of patients were low to intermediate risk per the D'Amico classification. Around 13% received upfront ADT. In total, 13 patients were treated with 35 Gy, and 10 were treated with 36.25 Gy. The rates of late > Grade 3 GU toxicity and > Grade 2 GI toxicity were 8.6% and 4.3%, respectively. There were no Grade 4 or 5 toxicities. Six patients (26%) developed a local recurrence at a median time of 7.5 years. Of these six patients, four had unilateral hip replacements and two had bilateral. Three underwent salvage cryotherapy and three received salvage ADT. Conclusions: In the general population, high-grade toxicities and local recurrences are uncommon following prostate SBRT. However, in this cohort of patients with prior hip replacements, prostate SBRT had higher than expected rates of late toxicity and local recurrence. In the opinion of the authors, such patients should be counseled regarding an elevated risk of late toxicity and local recurrence with prostate SBRT. With its ultrasound guidance, brachytherapy would have the advantage of circumventing the need for MRI/CT-based imaging and thus may represent a preferable radiation alternative in this patient population. If these patients are treated with SBRT, they should be monitored closely for local recurrence so early salvage can be performed. We hope that recent advances in metal artifact reduction techniques and dose -calculation algorithms will improve future outcomes.

Supplementary Fig. 5. Histone marks expression associated with resistance to sunitinib

Abstract Nonfunctioning macroadenoma is a commonly diagnosed pituitary tumor. Resection is the favored treatment, with radiosurgery often utilized for residual or progressing disease. Long-term outcomes are established for single-session radiosurgery, but mature outcomes are lacking for multisession radiosurgery. We report our institution’s 5-year efficacy and safety results for unfavorable nonfunctioning pituitary macroadenoma patients treated with 5-fraction robotic radiosurgery. Between 2010-2020, patients who completed 5-fraction radiosurgery for the treatment of unfavorable nonfunctioning pituitary macroadenomas were included. A tumor was considered unfavorable if the gross tumor volume (GTV) was larger than 5 cc or if it closely approached a critical structure (optic apparatus, brainstem or pituitary gland). Local control was calculated using the Kaplan-Meier Method. RESULTS Twenty predominately female patients (60%), age from 21-77 (median: 53 years) were included in this study. All underwent primary resection. Indications for radiosurgery included unresectable recurrence (85%) and residual disease progression (70%). Median tumor volume was 3.4 cm3 (range: 0.3-20.8 cm3) and 40% of the tumors were suprasellar. A mean dose of 30 Gy (range: 25Gy-30Gy), was delivered to a median isodose line of 80% (range: 75%-89%). Toxicity was minimal with 12 patients (40%) developing acute short-lived headaches and 1 patient (5%) developing a brief ipsilateral 6th nerve palsy. There was no radiation induced optic or pituitary dysfunction identified in this cohort. At a median follow up of 5 years local control at 5 years was 95%. There was 1 in-field failure pathologically confirmed following surgery for pituitary tumor hemorrhage and 2 radiographically confirmed out-of-field failures in patients with large tumors (> 20 cc) treated with lower doses (25 Gy). CONCLUSIONS The treatment of unfavorable nonfunctioning pituitary macroadenoma with 5-fraction robotic radiosurgery provides excellent local control to date, with minimal toxicity. Continued follow-up for the long term optic and pituitary function preservation and tumor control is needed.

Nonfunctioning macroadenoma is a commonly diagnosed pituitary tumor. Resection is the favored treatment, with radiosurgery often utilized for residual or progressing disease. Long-term outcomes are established for single-session radiosurgery, but mature outcomes are lacking for multisession radiosurgery. We report our institution’s 5-year efficacy and safety results for unfavorable nonfunctioning pituitary macroadenoma patients treated with 5-fraction robotic radiosurgery. Between 2010-2020, patients who completed 5-fraction radiosurgery for the treatment of unfavorable nonfunctioning pituitary macroadenomas were included. A tumor was considered unfavorable if the gross tumor volume (GTV) was larger than 5 cc or if it closely approached a critical structure (optic apparatus, brainstem or pituitary gland). Local control was calculated using the Kaplan-Meier Method. RESULTS Twenty predominately female patients (60%), age from 21-77 (median: 53 years) were included in this study. All underwent primary resection. Indications for radiosurgery included unresectable recurrence (85%) and residual disease progression (70%). Median tumor volume was 3.4 cm3 (range: 0.3-20.8 cm3) and 40% of the tumors were suprasellar. A mean dose of 30 Gy (range: 25Gy-30Gy), was delivered to a median isodose line of 80% (range: 75%-89%). Toxicity was minimal with 12 patients (40%) developing acute short-lived headaches and 1 patient (5%) developing a brief ipsilateral 6th nerve palsy. There was no radiation induced optic or pituitary dysfunction identified in this cohort. At a median follow up of 5 years local control at 5 years was 95%. There was 1 in-field failure pathologically confirmed following surgery for pituitary tumor hemorrhage and 2 radiographically confirmed out-of-field failures in patients with large tumors (> 20 cc) treated with lower doses (25 Gy). CONCLUSIONS The treatment of unfavorable nonfunctioning pituitary macroadenoma with 5-fraction robotic radiosurgery provides excellent local control to date, with minimal toxicity. Continued follow-up for the long term optic and pituitary function preservation and tumor control is needed.

We describe the utilization of SpaceOAR Vue™, a new iodinated rectal spacer, during Robotic Stereotactic Body Radiation Therapy (SBRT) for a Prostate Cancer Patient with a contraindication to Magnetic Resonance Imaging. A 69-year-old Caucasian male presented with unfavorable intermediate risk prostate cancer and elected to undergo SBRT. His medical history was significant for atrial fibrillation on Rivaroxaban with a pacemaker. He was felt to be at increased risk of radiation proctitis following SBRT due to the inability to accurately contour the anterior rectal wall at the prostate apex without a treatment planning MRI and an increased risk of late rectal bleeding due to prescribed anticoagulants. In this case report, we discuss the technical aspects of appropriate placement and treatment planning for utilizing SpaceOAR Vue™ with Robotic SBRT.

Background Usage of intraoperative indocyanine green (ICG) to assess skin flaps prior to abdominal wall closure has been shown to decrease postoperative wound-related complications. Primary outcome assessed is the utility of ICG in intraoperative decision making. Secondary outcomes analyzed are the incidence of surgical site occurrence (SSO) and hernia recurrence rates. Methods A retrospective study using the MedStar Georgetown University Hospital database was conducted, incorporating all consecutive patients undergoing complex incisional hernia repair from 2008 to 2018. 146 patients underwent perforator-sparing component separation (PSCST), 88 underwent flap assessment using intraoperative ICG angiography; they were then analyzed based on patient comorbidities, Ventral Hernia Working Group grade, operative factors, and complications. Results A total of 146 patients were analyzed with no statistical difference in patient characteristics between the SPY and no SPY group except in BMI (30.2 vs. 33.2 kg/m(2), p = 0.036). The no SPY group also had higher numbers of patients undergoing concurrent panniculectomy (12 vs. 1, p < 0.001), and extensive lysis of adhesions (30 vs. 31, p = 0.048). Of the 88 patients undergoing intraoperative SPY, 37 (42%) patients had a change of intraoperative management as defined by further subcutaneous skin flap debridement. Despite this change, there was no statistical difference in incidence of SSO between SPY and no SPY (24.3% vs. 11.8%, p = 0.12), and no difference in hernia recurrence rates 5.6% (n = 5) versus 13.7% (n = 8), p = 0.09. Conclusion Intraoperative ICG assessment of subcutaneous skin flaps with a perforator-sparing component separation does not result in a decrease in surgical site occurrences.

BACKGROUND:Class I histone deacetylases (HDACs) have been reported to be overexpressed in clear cell renal cell carcinoma (ccRCC), whereas the expression of class II HDACs is unknown.METHODS:Four isogenic cell lines C2/C2VHL and 786-O/786-OVHL with differential VHL expression are used in our studies. Cobalt chloride is used to mimic hypoxia in vitro. HIF-2α knockdowns in C2 and 786-O cells is used to evaluate the effect on HDAC 1 expression and activity. Invasion and migration assays are used to investigate the role of HDAC 1 and HDAC 6 expression in ccRCC cells. Comparisons are made between experimental groups using the paired T-test, the two-sample Student's T-test or one-way ANOVA, as appropriate. ccRCC and the TCGA dataset are used to observe the clinical correlation between HDAC 1 and HDAC 6 overexpression and overall and progression free survival.RESULTS:Our analysis of tumor and matched non-tumor tissues from radical nephrectomies showed overexpression of class I and II HDACs (HDAC6 only in a subset of patients). In vitro, both HDAC1 and HDAC6 over-expression increased cell invasion and motility, respectively, in ccRCC cells. HDAC1 regulated invasiveness by increasing matrix metalloproteinase (MMP) expression. Furthermore, hypoxia stimulation in VHL-reconstituted cell lines increased HIF isoforms and HDAC1 expression. Presence of hypoxia response elements in the HDAC1 promoter along with chromatin immunoprecipitation data suggests that HIF-2α is a transcriptional regulator of HDAC1 gene. Conversely, HDAC6 and estrogen receptor alpha (ERα) were co-localized in cytoplasm of ccRCC cells and HDAC6 enhanced cell motility by decreasing acetylated α-tubulin expression, and this biological effect was attenuated by either biochemical or pharmacological inhibition. Finally, analysis of human ccRCC specimens revealed positive correlation between HIF isoforms and HDAC. HDAC1 mRNA upregulation was associated with worse overall survival in the TCGA dataset.CONCLUSIONS:Taking together, these results suggest that HDAC1 and HDAC6 may play a role in ccRCC biology and could represent rational therapeutic targets.

Abstract Sunitinib is considered a first-line therapeutic option for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite sunitinib's clinical efficacy, patients eventually develop drug resistance and disease progression. Herein, we tested the hypothesis whether initial sunitinib resistance may be transient and could be overcome by dose increase. In selected patients initially treated with 50 mg sunitinib and presenting with minimal toxicities, sunitinib dose was escalated to 62.5 mg and/or 75 mg at the time of tumor progression. Mice bearing two different patient-derived ccRCC xenografts (PDX) were treated 5 days per week with a dose-escalation schema (40–60–80 mg/kg sunitinib). Tumor tissues were collected before dose increments for immunohistochemistry analyses and drug levels. Selected intrapatient sunitinib dose escalation was safe and several patients had added progression-free survival. In parallel, our preclinical results showed that PDXs, although initially responsive to sunitinib at 40 mg/kg, eventually developed resistance. When the dose was incrementally increased, again we observed tumor response to sunitinib. A resistant phenotype was associated with transient increase of tumor vasculature despite intratumor sunitinib accumulation at higher dose. In addition, we observed associated changes in the expression of the methyltransferase EZH2 and histone marks at the time of resistance. Furthermore, specific EZH2 inhibition resulted in increased in vitro antitumor effect of sunitinib. Overall, our results suggest that initial sunitinib-induced resistance may be overcome, in part, by increasing the dose, and highlight the potential role of epigenetic changes associated with sunitinib resistance that can represent new targets for therapeutic intervention. Mol Cancer Ther; 14(2); 513–22. ©2014 AACR.

Abstract Background: Sunitinib is first-line therapy for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite the clinical efficacy of sunitinib, in the majority of patients the disease eventually develops resistance and progresses. Epigenetic modifications of histone protein in chromatins have been shown to play a role in the regulation of gene transcription patterns in cells primarily by the catalytic activity of histone methyltransferase. EZH2 has been shown to contribute to tumor angiogenesis by inactivating anti-angiogenic factors via methylation at their promoter region. In this study, we examined epigenetic changes that may be associated with response and the resistant phenotype. Methods: Human ccRCC xenograft models RP-01 (isolated from a skin metastasis in a patient with sporadic ccRCC) and RP-02 (isolated from a skin metastasis in a patient with hereditary ccRCC) were implanted (∼1mm pieces) subcutaneously into SCID mice and were randomly assigned into two groups (sunitinib and vehicle). Mice were treated 5 days/week with sunitinib at 40mg/kg. Tumor volumes and body weights were assessed weekly by caliper measurements and weigh scale, respectively. Tumor tissues and blood were collected prior treatments and when tumors became resistant to treatment. Tissues collected were used for immunohistochemistry studies and RNA analysis. Results: Our results showed that RP-01 and RP-02 tumors, although initially responsive to treatment at 40 mg/kg, eventually became resistant to treatment. Immunohistochemistry analysis of RP-01 tumor samples indicated tumors to be hypo-vasculature when responding to sunitinib, but then became hyper-vascularized at point of resistance by CD31 staining. Analysis also revealed an associated increase in the expression of the methyltransferase EZH2 when tumors became resistant to sunitinib. However, there was no change in the expression levels of its EZH2 associated histone make H3K27me3. In addition, we noticed an increase in the levels of H3k9me2 and H3k4me2 in tumors resistant to sunitinib as compared to controls. Conclusion: Overall, our results suggest the potential role of epigenetic changes that that may be associated with sunitinib-induced resistance in ccRCC Citation Format: REMI ADELAIYE, Kiersten Marie Miles, Eric Ciamporcero, Dylan Conroy, Swathi Ramakrishnan, Ashley Orillion, Sheng Yu Ku, May Elbanna, Li shen, Sreenivasulu Chintala, Roberto Pili. Epigenetic changes associated with resistance to sunitinib in human clear cell renal cell carcinoma models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1375. doi:10.1158/1538-7445.AM2014-1375

BACKGROUND:The Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis. Blockade of the Dll4-Notch pathway in preclinical cancer models has been associated with non-productive angiogenesis and reduced tumor growth. Given the cross-talk between the vascular endothelial growth factor (VEGF) and Delta-Notch pathways in tumor angiogenesis, we examined the activity of a function-blocking Dll4 antibody, REGN1035, alone and in combination with anti-VEGF therapy in renal cell carcinoma (RCC). METHODS AND RESULTS:Severe combined immunodeficiency (SCID) mice bearing patient-derived clear cell RCC xenografts were treated with REGN1035 and in combination with the multi-targeted tyrosine kinase inhibitor sunitinib or the VEGF blocker ziv-aflibercept. Immunohistochemical and immunofluorescent analyses were carried out, as well as magnetic resonance imaging (MRI) examinations pre and 24 hours and 2 weeks post treatment. Single agent treatment with REGN1035 resulted in significant tumor growth inhibition (36-62%) that was equivalent to or exceeded the single agent anti-tumor activity of the VEGF pathway inhibitors sunitinib (38-54%) and ziv-aflibercept (46%). Importantly, combination treatments with REGN1035 plus VEGF inhibitors resulted in enhanced anti-tumor effects (72-80% growth inhibition), including some tumor regression. Magnetic resonance imaging showed a marked decrease in tumor perfusion in all treatment groups. Interestingly, anti-tumor efficacy of the combination of REGN1035 and ziv-aflibercept was also observed in a sunitinib resistant ccRCC model. CONCLUSIONS:Overall, these findings demonstrate the potent anti-tumor activity of Dll4 blockade in RCC patient-derived tumors and a combination benefit for the simultaneous targeting of the Dll4 and VEGF signaling pathways, highlighting the therapeutic potential of this treatment modality in RCC.

Abstract Introduction: Histone deacetylases are overexpressed in several tumors including prostate, breast and clear cell renal cell carcinoma (ccRCC). Our group has previously reported that class II HDACs, HDAC4 and HDAC6 regulate HIF-α stability in ccRCC cell lines. Interestingly, HDAC6 overexpression in ER-α positive breast cancer has been shown to correlate with overall and cancer specific survival in response to tamoxifen treatment. In addition, HDAC6 increases cell motility by deacetylating α-tubulin, and HDAC6 interaction with ER-α on the cell membrane increases its deacetylating activity. The objective of our study was to assess whether HDAC6 associates with ER-α in a subset of ccRCC and whether this association can be targeted therapeutically. Methods: Radical nephrectomy tumor samples (n=14) with matched adjacent non tumor tissues were collected and analyzed for HDAC6 expression by Western blot analysis. HDAC6 expression was also assessed in C2, C2VHL and 786-O ccRCC cell lines by Western blot and immunofluorescence analysis. HDAC6 and ER-α colocalization was examined by immunoprecipitation and immunofluorescence. HDAC 6 was overexpressed in cell lines and investigated for cell motility by scratch assay. Cell lines were also treated with hydroxy tamoxifen in short term (4 hours) and long term (24, 48 and 72 hours) culture experiments for evaluation of effects on acetylated α-tubulin and cell proliferation, respectively. Results: Analysis of matched patient tumor samples revealed that a subset of ccRCC had higher HDAC6 expression as compared to the adjacent non tumor tissue. HDAC6 and ER-α examined in ccRCC tumors (n=44) by immunofluorescence showed overexpression in 10% of tumor samples. Immunoprecipitation of HDAC 6 in ccRCC cell lines showed that ER-α is present in the same complex as HDAC 6 as confirmed also by fluorescence microscopy. HDAC6 overexpression in ccRCC cell lines increased cell motility, although overexpression did not affect cell proliferation. Cells treated for short term experiments with hydroxy tamoxifen showed an increase in acetylated α-tubulin when examined by immunofluorescence. Upon long term hydroxy tamoxifen treatment in regular DMEM medium with serum, ccRCC cell proliferation was affected at high concentrations (10-20µM), similar to MCF 7 cells treated under similar conditions. Conclusions: HDAC 6 and ER-α are overexpressed in a subset of ccRCC. HDAC6 overexpression affects cell motility but not proliferation. HDAC6 and ER-α are present in the same immunocomplex and this association may be targeted with therapeutic interventions. Ongoing studies are testing concomitant, either genetic or pharmacological, inhibition of both HDAC6 and ER-α in ccRCC and will provide the rationale for novel targeted therapies for a selected group of patients with ccRCC. Citation Format: Swathi Ramakrishnan, Sheng-Yu Ku, Wendy Swetzig, Dylan Conroy, Li Shen, Sreenivasulu Chintala, Paula Sotomayor, Kiersten M. Miles, Remi Adelaiye, Eric Ciamporcero, Ashley Orillion, Leigh Ellis, Gokul Das, Roberto Pili. Evidence for hdac6 and er-α association in a subset of clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4061. doi:10.1158/1538-7445.AM2014-4061