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To compare the toxicity and treatment outcomes in human immunodeficiency virus (HIV)-positive versus HIV-negative patients with squamous cell carcinoma of the anal canal who underwent definitive concurrent chemoradiation at a single institution.Fifty-three consecutive HIV-positive patients treated between 1987 and 2013 were compared with 205 consecutive HIV-negative patients treated between 2003 and 2013. All patients received radiotherapy at a single regional facility. The median radiation dose was 54 Gy (range, 28 to 60 Gy). Concurrent chemotherapy consisted of 2 cycles 5-FU with mitomycin-C given on day 1±day 29). After treatment, patients were closely followed with imaging studies, clinical examinations, and rigid proctoscopies. Outcomes assessed were toxicity rates, progression-free survival, colostomy-free survival, cancer-specific survival, and overall survival.Median follow-up was 34 months. Compared with HIV-negative patients, HIV-positive patients were younger (median age, 48 vs. 62 y) and predominantly male sex (98% of HIV-positive patients were male vs. 22% of HIV-negative patients). Of the HIV-positive patients, 37 (70%) were on highly active antiretroviral therapy, 26 (65%) had an undetectable viral load at the time of treatment, and 36 (72%) had a CD4 count>200 (mean CD4 count, 455). There were no significant differences in acute or late nonhematologic or hematologic toxicity rates between the 2 groups. At 3 years, there was no significant difference between HIV-positive and HIV-negative patients in regards to progression-free survival (75% vs. 76%), colostomy-free survival (85% vs. 85%), or cancer-specific survival (79% vs. 88%, P=0.36), respectively. On univariate analysis, there was a trend toward worse overall survival in HIV-positive patients (72% vs. 84% at 3 y, P=0.06). For the entire cohort, on multivariate analysis only male sex and stage were predictive of worse survival outcomes. HIV status was not associated with worse outcomes in Cox models.In the highly active antiretroviral therapy era, HIV-positive patients with anal cancer treated with standard definitive chemoradiation have equivalent toxicity and cancer-specific survival compared with HIV-negative patients.