- Email: [email protected]
Immunohistochemical Verification of Stereotactic Proton Radiosurgery Targeting Accuracy in the Rat Brain
I. J. Radiation Oncology d Biology d Physics
S662
Volume 78, Number 3, Supplement, 2010
Results: We found APC hypermethylation in sera of 29% (25/85), RASSF1A in 26% (22/85), GSTP1 in 18% (14/76) and ESR1 38% (32/85) of breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were found CTC positive by detecting EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of positives were found for methylated RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive HER2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above parameters. The presence of CTC in peripheral blood was significantly associated with the methylated APC (p = 0.012) as well as methylated GSTP1 (p = 0.001). Conclusions: The detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC did not show exclusive specifity but were a phenotypic feature of more aggressive tumor biology and advanced disease. Author Disclosure: E. Boelke, None; C. Matuschek, None; M. Peiper, None; H. Prisack, None; A. Gerber, None; W. Budach, None; H. Bojar, None.
3027
Gene Expression Differences of Peripheral Blood Cells Predict Pathological Complete Response to Neoadjuvant Concurrent Chemoradiation in Patients with Esophageal Squamous Cell Carcinoma
F. Hsu1,2, E. Y. Chuang2, Y. Lee3, J. Lee3, C. Hsu1,4, C. Lin1,4, Y. Tsai1,4, J. C. Cheng1,5 1 Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan, 2Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan, 3Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan, 4Cancer Research Center, National Taiwan University College of Medicine, Taipei, Taiwan, 5Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan
Purpose/Objective(s): This prospective study analyzed gene expression differences of peripheral blood cells in predicting pathological response to neoadjuvant concurrent chemoradiation (CCRT) for esophageal squamous cell carcinoma. Materials/Methods: Twenty-one patients with esophageal squamous cell carcinoma completed the whole course of neoadjuvant CCRT followed by subsequent radical esophagectomy were enrolled for analysis. Patients received radiotherapy with 40 Gy in 20 fractions and concurrent chemotherapy with 5-fluorouracil plus cisplatin or paclitaxel/docetaxel plus cisplatin. Venous peripheral blood was obtained before and after CCRT. Gene expression profiles on microarray chips were analyzed and correlated to pathological treatment response. Results: Of 21 patients, 10 had pathological complete response (pCR). Using significance analysis of microarrays, 343 genes were significantly influenced after CCRT. The responses of 17 genes were correlated with pCR by Bayesian analysis of variance for microarrays. Wilcoxon rank test was then used to control type 1 error and identify 10 candidate genes associated with pCR (p value \ 0.01). These genes included AK3L1, C3orf64, CCNL1, FAM13A1, FAM84B, GYPC, HIST2H4A, NARG2, SEPT4, and TPD52. Conclusions: This study found that gene expression differences of peripheral blood cells might associate with pCR in patients with esophageal squamous cell carcinoma undergoing neoadjuvant CCRT. Further validation is required to investigate the potentials of these genes as the biomarkers predicting treatment response. Author Disclosure: F. Hsu, None; E.Y. Chuang, Grant NCTRC200730 from the National Center of Excellence for Clinical Trial and Research, Taiwan, B. Research Grant; Y. Lee, None; J. Lee, None; C. Hsu, None; C. Lin, None; Y. Tsai, None; J.C. Cheng, Grant NCTRC200730 from the National Center of Excellence for Clinical Trial and Research, Taiwan, B. Research Grant.
3028
Immunohistochemical Verification of Stereotactic Proton Radiosurgery Targeting Accuracy in the Rat Brain
Y. Nie Loma Linda University, Loma Linda, CA Purpose/Objective(s): We have developed a system for targeting functional areas in the rat brain with narrow proton beams. Due to the extremely sharp lateral dose falloff of high-energy proton beams, a cross-fire technique with multiple shoot-through beams converging on the target will be used to selectively irradiate small regions in the rat brain. The spatial accuracy of dose delivery is crucial and needs to be verified. This ongoing study intends to verify the accuracy and precision of the targeting method by developing immunohistochemical staining protocols based on IgG leakage through the radiation-induced breakdown of the blood-brain barrier (BBB) and various DNA damage markers. Materials/Methods: Male Sprague-Dawley (8 weeks, 150-190g) rats were subjected to hemispheric proton irradiation with 250 MeV proton beams at doses of 0 Gy, 8 Gy, and 25 Gy. IgG extravasation was used to show BBB damage from 4 to 72 hours after irradiation. Rat brains perfused with 4% paraformaldehyde-perfused were cryostat-sectioned and slide-mounted. Sections then underwent immunohistochemical staining using the ABC method (Santa Cruz Biotechnology). Radiation-induced H2AX phosphorylation, a sensitive marker of DNA double strand breaks (DSBs), was detected by Donkey anti-mouse IgG Alexa Fluor 488 (green), while nuclei were counterstained with propidium iodide (PI, red). Results: After 25 Gy proton irradiation, radiation-induced BBB damage resulted in IgG extravasation as indicated by positive immunostaining for anti-IgG. Detection of gH2AX was also possible after hemibrain proton irradiation with 8 Gy.
Proceedings of the 52nd Annual ASTRO Meeting Conclusions: IgG staining is sensitive to local vascular radiation damage, which has the advantage of being maintained during longer time intervals (days to weeks rather than hours) compared to short-term DNA damage markers. The DSB marker gH2AX can be used for rapid confirmation of the targeted area. IgG staining and gH2AX immunofluorescences are useful for microscopic verification of the targeting accuracy of small-animal radiosurgery procedures with sub-millimeter resolution. Author Disclosure: Y. Nie, None.
3029
Intracranial Murine Tumor Investigation of Radiation and Antiangiogenic Agents using Serial MRI
1
C. Chung , W. Foltz1, P. Lindsay1, K. Burrell2, J. Kallehauge1, P. Wildgoose1, K. Camphausen3, D. Jaffray1, G. Zadeh4, C. Menard1 1 PMH, Toronto, ON, Canada, 2SickKids Hospital, Toronto, ON, Canada, 3ROB/NCI, Bethesda, MD, 4Toronto Western Hospital, Toronto, ON, Canada
Purpose/Objective(s): This study aims to investigate the feasibility and utility of serial magnetic resonance imaging (MRI) to guide the design, delivery and evaluation of a murine intracranial (IC) tumor experiment of radiation (RT) and anti-angiogenic (AA) therapy. Materials/Methods: U87 glioma cells were injected IC into 58 NOD SCID mice. Serial MRI with gadolinium via tail vein were acquired at day 0 (7days post-IC), and days 3,7,10,14 and 21. Mice with MRI visible tumors at day 0 were stratified by tumor size to:(1) placebo (CTRL) (2) RT+placebo (RT) (3) Sunitinib (SU) (4) RT+SU (SURT). Day 0 MRI tumor-targeted and conebeam CT (CBCT) guided RT was used to deliver 8Gy on day 1. SU 0.8mg or placebo was given via oral gavage for 7 days starting day 1. MRI evaluation included: (1) T2-weighted (2) Quantitative T1 (3) Dynamic contrast-enhanced (DCE) (4) Diffusion weighted (DWI) (5) T1-weighted with gadolinium. RT dose to each tumor was evaluated on fused images of CBCT and day 0 MRI. Results: Fifty-two mice with visible tumors were assigned to treatment arms to ensure similar baseline tumor volumes for all arms: 0.60+0.04mm3 [Range: 0.09-1.6mm3]. No mice expired with serial imaging. All irradiated tumors received .90% prescribed dose. Tumor growth rate was better for SU+RT than placebo (p\0.001) or SU (p = 0.003) but was similar between RT and SU+RT (p = 0.4). By day 3, DCE (iAUC60) decreased in SU+RT 31+7% but increased in CTRL16+1% (p = 0.00009), RT 34+16% (p = 0.0008), and SU15+8% (p = 0.0004). Rise from baseline in %ADC(tumor/normal brain) became significantly greater by day10 for SURT and RT arms (22%, p = 0.01) but not for SU (12%, p = 0.9) vs. CTRL 11%. Conclusions: This study demonstrates serial MRI studies are feasible and augments investigation of RT and AA in murine IC tumor models by providing temporal, spatial and physiological information. Serial MRI allowed identification of promising biomarkers and their optimal timing: statistically significant decline in iAUC60 at day 3 and rise in ADC by day 10. This data will guide future preclinical studies to evaluate the underlying pathophysiology and timing of clinical imaging for biomarker evaluations. Author Disclosure: C. Chung, Received Sunitinib Drug from Pfizer for the purpose of this pre-clinical study, C. Other Research Support; W. Foltz, None; P. Lindsay, None; K. Burrell, None; J. Kallehauge, None; P. Wildgoose, None; K. Camphausen, None; D. Jaffray, None; G. Zadeh, None; C. Menard, Received Sunitinib Drug from Pfizer for the purpose of this pre-clinical study, C. Other Research Support.
3030
Role of Excision Repair Cross Complementation 1 Expression as a Prognostic Marker for Response to Radiotherapy in Early Stage Laryngeal Cancer
K. L. Johung1, A. Rewari2, H. Wu2, J. Contessa1, B. Haffty2, R. Decker1 1 Yale University School of Medicine, New Haven, CT, 2The Cancer Institute of New Jersey, UMDNJ/Robert Wood Johnson Medical School, New Brunswick, NJ
Purpose/Objective(s): The excision repair cross complementation 1 (ERCC1) endonuclease is critical for a rate-limiting step in the nucleotide excision repair pathway. High levels of ERCC1 mRNA and protein expression have been shown to correlate with resistance to platinum-based chemotherapy or concurrent chemoradiotherapy, as well as inferior progression free survival and overall survival in patients with primary lung cancers or cancers of the head and neck. In addition, single nucleotide polymorphisms in the ERCC1 gene are predictive of response to radiotherapy in patients with early stage squamous cell carcinoma of the head and neck. Therefore tissue microarray analysis was used to determine the prognostic value of ERCC1 expression in a cohort of early stage laryngeal cancer treated with radiotherapy alone. Materials/Methods: Tissue microarrays were constructed from the pre-treatment biopsies of 123 patients with stages I and II squamous cell carcinoma of the larynx treated between 1975 and 2000 with radiation therapy to a median dose of 66Gy using standard fields and fractionation. 84 (68.3%) of the cases were T1, and 39 (31.7%) were T2. With a median follow-up of 9.9 years, the 5 year local recurrence free rate was 70.4% and the 5 year overall survival was 60.2% for the entire cohort. Immunohistochemistry was used to determine ERCC1 expression. Results: Expression could be analyzed in 90 patients (73%) and positive expression was detected in 48 of the 90 cases (53%). Bivariate analysis showed that increased ERCC1 expression was associated with lower T stage (p \ 0.04), with 60% of T1 tumors positive for ERCC1 expression compared to only 36% of T2 tumors. Other prognostic factors including age .60, race, sex, and tumor subsite (supraglottic vs. glottic) did not significantly correlate with ERCC1 expression. Interestingly, univariate survival analysis showed that ERCC1 expression does not significantly correlate with locoregional recurrence free survival or overall survival at 5 and 10 years, though there was a non-significant trend towards worse outcome with ERCC1 expression. Overall survival at 5 and 10 years was 77.7% vs. 55.8% (p = 0.12) and 64.9% vs. 33.0% (p = 0.08), and local
S663
S662
Volume 78, Number 3, Supplement, 2010
Results: We found APC hypermethylation in sera of 29% (25/85), RASSF1A in 26% (22/85), GSTP1 in 18% (14/76) and ESR1 38% (32/85) of breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were found CTC positive by detecting EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of positives were found for methylated RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive HER2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above parameters. The presence of CTC in peripheral blood was significantly associated with the methylated APC (p = 0.012) as well as methylated GSTP1 (p = 0.001). Conclusions: The detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC did not show exclusive specifity but were a phenotypic feature of more aggressive tumor biology and advanced disease. Author Disclosure: E. Boelke, None; C. Matuschek, None; M. Peiper, None; H. Prisack, None; A. Gerber, None; W. Budach, None; H. Bojar, None.
3027
Gene Expression Differences of Peripheral Blood Cells Predict Pathological Complete Response to Neoadjuvant Concurrent Chemoradiation in Patients with Esophageal Squamous Cell Carcinoma
F. Hsu1,2, E. Y. Chuang2, Y. Lee3, J. Lee3, C. Hsu1,4, C. Lin1,4, Y. Tsai1,4, J. C. Cheng1,5 1 Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan, 2Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan, 3Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan, 4Cancer Research Center, National Taiwan University College of Medicine, Taipei, Taiwan, 5Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan
Purpose/Objective(s): This prospective study analyzed gene expression differences of peripheral blood cells in predicting pathological response to neoadjuvant concurrent chemoradiation (CCRT) for esophageal squamous cell carcinoma. Materials/Methods: Twenty-one patients with esophageal squamous cell carcinoma completed the whole course of neoadjuvant CCRT followed by subsequent radical esophagectomy were enrolled for analysis. Patients received radiotherapy with 40 Gy in 20 fractions and concurrent chemotherapy with 5-fluorouracil plus cisplatin or paclitaxel/docetaxel plus cisplatin. Venous peripheral blood was obtained before and after CCRT. Gene expression profiles on microarray chips were analyzed and correlated to pathological treatment response. Results: Of 21 patients, 10 had pathological complete response (pCR). Using significance analysis of microarrays, 343 genes were significantly influenced after CCRT. The responses of 17 genes were correlated with pCR by Bayesian analysis of variance for microarrays. Wilcoxon rank test was then used to control type 1 error and identify 10 candidate genes associated with pCR (p value \ 0.01). These genes included AK3L1, C3orf64, CCNL1, FAM13A1, FAM84B, GYPC, HIST2H4A, NARG2, SEPT4, and TPD52. Conclusions: This study found that gene expression differences of peripheral blood cells might associate with pCR in patients with esophageal squamous cell carcinoma undergoing neoadjuvant CCRT. Further validation is required to investigate the potentials of these genes as the biomarkers predicting treatment response. Author Disclosure: F. Hsu, None; E.Y. Chuang, Grant NCTRC200730 from the National Center of Excellence for Clinical Trial and Research, Taiwan, B. Research Grant; Y. Lee, None; J. Lee, None; C. Hsu, None; C. Lin, None; Y. Tsai, None; J.C. Cheng, Grant NCTRC200730 from the National Center of Excellence for Clinical Trial and Research, Taiwan, B. Research Grant.
3028
Immunohistochemical Verification of Stereotactic Proton Radiosurgery Targeting Accuracy in the Rat Brain
Y. Nie Loma Linda University, Loma Linda, CA Purpose/Objective(s): We have developed a system for targeting functional areas in the rat brain with narrow proton beams. Due to the extremely sharp lateral dose falloff of high-energy proton beams, a cross-fire technique with multiple shoot-through beams converging on the target will be used to selectively irradiate small regions in the rat brain. The spatial accuracy of dose delivery is crucial and needs to be verified. This ongoing study intends to verify the accuracy and precision of the targeting method by developing immunohistochemical staining protocols based on IgG leakage through the radiation-induced breakdown of the blood-brain barrier (BBB) and various DNA damage markers. Materials/Methods: Male Sprague-Dawley (8 weeks, 150-190g) rats were subjected to hemispheric proton irradiation with 250 MeV proton beams at doses of 0 Gy, 8 Gy, and 25 Gy. IgG extravasation was used to show BBB damage from 4 to 72 hours after irradiation. Rat brains perfused with 4% paraformaldehyde-perfused were cryostat-sectioned and slide-mounted. Sections then underwent immunohistochemical staining using the ABC method (Santa Cruz Biotechnology). Radiation-induced H2AX phosphorylation, a sensitive marker of DNA double strand breaks (DSBs), was detected by Donkey anti-mouse IgG Alexa Fluor 488 (green), while nuclei were counterstained with propidium iodide (PI, red). Results: After 25 Gy proton irradiation, radiation-induced BBB damage resulted in IgG extravasation as indicated by positive immunostaining for anti-IgG. Detection of gH2AX was also possible after hemibrain proton irradiation with 8 Gy.
Proceedings of the 52nd Annual ASTRO Meeting Conclusions: IgG staining is sensitive to local vascular radiation damage, which has the advantage of being maintained during longer time intervals (days to weeks rather than hours) compared to short-term DNA damage markers. The DSB marker gH2AX can be used for rapid confirmation of the targeted area. IgG staining and gH2AX immunofluorescences are useful for microscopic verification of the targeting accuracy of small-animal radiosurgery procedures with sub-millimeter resolution. Author Disclosure: Y. Nie, None.
3029
Intracranial Murine Tumor Investigation of Radiation and Antiangiogenic Agents using Serial MRI
1
C. Chung , W. Foltz1, P. Lindsay1, K. Burrell2, J. Kallehauge1, P. Wildgoose1, K. Camphausen3, D. Jaffray1, G. Zadeh4, C. Menard1 1 PMH, Toronto, ON, Canada, 2SickKids Hospital, Toronto, ON, Canada, 3ROB/NCI, Bethesda, MD, 4Toronto Western Hospital, Toronto, ON, Canada
Purpose/Objective(s): This study aims to investigate the feasibility and utility of serial magnetic resonance imaging (MRI) to guide the design, delivery and evaluation of a murine intracranial (IC) tumor experiment of radiation (RT) and anti-angiogenic (AA) therapy. Materials/Methods: U87 glioma cells were injected IC into 58 NOD SCID mice. Serial MRI with gadolinium via tail vein were acquired at day 0 (7days post-IC), and days 3,7,10,14 and 21. Mice with MRI visible tumors at day 0 were stratified by tumor size to:(1) placebo (CTRL) (2) RT+placebo (RT) (3) Sunitinib (SU) (4) RT+SU (SURT). Day 0 MRI tumor-targeted and conebeam CT (CBCT) guided RT was used to deliver 8Gy on day 1. SU 0.8mg or placebo was given via oral gavage for 7 days starting day 1. MRI evaluation included: (1) T2-weighted (2) Quantitative T1 (3) Dynamic contrast-enhanced (DCE) (4) Diffusion weighted (DWI) (5) T1-weighted with gadolinium. RT dose to each tumor was evaluated on fused images of CBCT and day 0 MRI. Results: Fifty-two mice with visible tumors were assigned to treatment arms to ensure similar baseline tumor volumes for all arms: 0.60+0.04mm3 [Range: 0.09-1.6mm3]. No mice expired with serial imaging. All irradiated tumors received .90% prescribed dose. Tumor growth rate was better for SU+RT than placebo (p\0.001) or SU (p = 0.003) but was similar between RT and SU+RT (p = 0.4). By day 3, DCE (iAUC60) decreased in SU+RT 31+7% but increased in CTRL16+1% (p = 0.00009), RT 34+16% (p = 0.0008), and SU15+8% (p = 0.0004). Rise from baseline in %ADC(tumor/normal brain) became significantly greater by day10 for SURT and RT arms (22%, p = 0.01) but not for SU (12%, p = 0.9) vs. CTRL 11%. Conclusions: This study demonstrates serial MRI studies are feasible and augments investigation of RT and AA in murine IC tumor models by providing temporal, spatial and physiological information. Serial MRI allowed identification of promising biomarkers and their optimal timing: statistically significant decline in iAUC60 at day 3 and rise in ADC by day 10. This data will guide future preclinical studies to evaluate the underlying pathophysiology and timing of clinical imaging for biomarker evaluations. Author Disclosure: C. Chung, Received Sunitinib Drug from Pfizer for the purpose of this pre-clinical study, C. Other Research Support; W. Foltz, None; P. Lindsay, None; K. Burrell, None; J. Kallehauge, None; P. Wildgoose, None; K. Camphausen, None; D. Jaffray, None; G. Zadeh, None; C. Menard, Received Sunitinib Drug from Pfizer for the purpose of this pre-clinical study, C. Other Research Support.
3030
Role of Excision Repair Cross Complementation 1 Expression as a Prognostic Marker for Response to Radiotherapy in Early Stage Laryngeal Cancer
K. L. Johung1, A. Rewari2, H. Wu2, J. Contessa1, B. Haffty2, R. Decker1 1 Yale University School of Medicine, New Haven, CT, 2The Cancer Institute of New Jersey, UMDNJ/Robert Wood Johnson Medical School, New Brunswick, NJ
Purpose/Objective(s): The excision repair cross complementation 1 (ERCC1) endonuclease is critical for a rate-limiting step in the nucleotide excision repair pathway. High levels of ERCC1 mRNA and protein expression have been shown to correlate with resistance to platinum-based chemotherapy or concurrent chemoradiotherapy, as well as inferior progression free survival and overall survival in patients with primary lung cancers or cancers of the head and neck. In addition, single nucleotide polymorphisms in the ERCC1 gene are predictive of response to radiotherapy in patients with early stage squamous cell carcinoma of the head and neck. Therefore tissue microarray analysis was used to determine the prognostic value of ERCC1 expression in a cohort of early stage laryngeal cancer treated with radiotherapy alone. Materials/Methods: Tissue microarrays were constructed from the pre-treatment biopsies of 123 patients with stages I and II squamous cell carcinoma of the larynx treated between 1975 and 2000 with radiation therapy to a median dose of 66Gy using standard fields and fractionation. 84 (68.3%) of the cases were T1, and 39 (31.7%) were T2. With a median follow-up of 9.9 years, the 5 year local recurrence free rate was 70.4% and the 5 year overall survival was 60.2% for the entire cohort. Immunohistochemistry was used to determine ERCC1 expression. Results: Expression could be analyzed in 90 patients (73%) and positive expression was detected in 48 of the 90 cases (53%). Bivariate analysis showed that increased ERCC1 expression was associated with lower T stage (p \ 0.04), with 60% of T1 tumors positive for ERCC1 expression compared to only 36% of T2 tumors. Other prognostic factors including age .60, race, sex, and tumor subsite (supraglottic vs. glottic) did not significantly correlate with ERCC1 expression. Interestingly, univariate survival analysis showed that ERCC1 expression does not significantly correlate with locoregional recurrence free survival or overall survival at 5 and 10 years, though there was a non-significant trend towards worse outcome with ERCC1 expression. Overall survival at 5 and 10 years was 77.7% vs. 55.8% (p = 0.12) and 64.9% vs. 33.0% (p = 0.08), and local
S663