This is a 20 patient pilot study to examine the feasibility of dose-adapted radiation therapy for the treatment of locally advanced anal squamous cell cancer. The tumor and a patient's anatomy may change during radiation treatment and daily adaption of the radiation plan (i.e., a new daily plan based on the anatomy of the day) may help to maximize the dose to the tumor and minimize the radiation dose to the normal surrounding organs.

The standard treatment for Human Papilloma Virus (HPV)-positive locally advanced anal cancer (described as a tumor that is greater than 4 cm in size, or with positive lymph nodes) is 54 Gy of radiation treatment to the anal canal and primary tumor planning total volume (PTV), 50.4-54 Gy to positive nodal PTV and 45 Gy to elective lymph node PTV with 5-fluorouracil (5-FU) and mitomycin-C chemotherapy administered at the same time as radiation in 30 fraction (treatments) delivery. During the six week course of radiotherapy, there is often a notable decrease in volume of the tumor (both primary and regional nodes), as early as one week into treatment, detected on weekly on-board Cone Beam Computed Tomography (CBCT), which is a scan done on the treatment machine while patients receive radiation to ensure that the tumor is being treated and normal tissue is not. However, CT simulation (a CT scan used to plan radiation treatment) and re-planning of the treatment to account for the tumor shrinkage are not routinely performed due to time, patient inconvenience and staffing resources. As such, daily adaptive radiation, which can generate a new CT-based plan using the anatomy of the day, may be a time efficient method to both plan and treat the patient.

Inclusion Criteria:

• Histologically proven, invasive primary squamous, basaloid or cloacogenic carcinoma of the anal canal.
• American Joint Committee on Cancer (AJCC) 8th edition stage T2 > 4 cm, T3-4 or N1.
• Age ≥18 years.
• Eastern Cooperative Oncology Group (ECOG) performance status ≤2 (Karnofsky ≥60%).
• Life expectancy of greater than 12 months.
• Patients must have normal organ and marrow function as defined below:
• leukocytes greater than or equal to 3,000/microliter
• absolute neutrophil count greater than or equal to 1,500/microliter
• platelets greater than or equal to 100,000/microliter
• total bilirubin within normal institutional limits
• Aspartate transaminase (AST)(SGOT)/Alanine transaminase (ALT)(SGPT) ≤ 2.5 × institutional upper limit of normal
• creatinine within normal institutional limits OR creatinine clearance ≥60 mL/min/1.73 m2 for patients with creatinine levels above institutional normal.
• Females of childbearing potential and males must agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry, for the duration of study participation, and for 6 months after completion of study therapy. All pregnancies must be reported.
• Ability to understand and the willingness to sign a written informed consent document.

Exclusion Criteria:

• Prior or co-existing invasive malignancy (except non-melanomatous skin cancer) unless disease free ≥ 2 years.
• Prior chemotherapy or radiation for anal cancer.
• Patients who have undergone complete surgical resection.
• Presence of recurrent/metastatic disease.
• Prior allergic reaction to 5-Fluorouracil or mitomycin C.
• Artificial organ prosthetics, pacemakers or other implantable devices.
• Prior radiotherapy to the pelvis that would result in overlap of radiation therapy fields.
• Uncontrolled inter-current illness including but not limited to known history of HIV with cluster of differentiation 4 (CD4) count less than 200 or symptomatic cardiac disease.
• Women who are pregnant or lactating.