This clinical trial proposes to evaluate a relatively unexplored approach to treatment of squamous cell carcinoma (SCC) on the lower extremities. The strategy is to directly and specifically deliver drug to the tumor. For the proposed phase I clinical trial, the investigators will perform intralesional injections of a well characterized, potent chemotherapeutic agent 5-fluorouracil (5FU) with and without a topical application of 0.005% calcipotriene cream to kill topically accessible SCC cells. The goal of the study is to evaluate the safety profile and tolerability of intralesional-5FU with and without a concomitant topical calcipotriene and measure the clinical objective response rate (ORR) in treated lesions compared to untreated lesions 3 weeks after treatment.

Squamous cell carcinoma of the skin is a common form of keratinocyte skin cancer. The majority of cutaneous SCCs occur on the head and neck, more so in men than women, but can occur anywhere squamous cells are found. Squamous cell carcinoma of the skin is usually not life-threatening, though it can be aggressive in some cases, and is normally treated with minor surgery.

Squamous cell carcinoma (SCC) of the lower extremity is a distinct subset of cutaneous squamous cell carcinomas which tend to occur multiply in elderly women. Histopathological studies of lower extremity SCCs reveals that they tend to be well differentiated and have low incidence of perineural and lymphovascular invasion and are also less prone to metastasis. Minor surgery has been the standard of care for this subset of SCC but leads to many complications such as poor wound healing and postoperative infections. Furthermore, a phenomenon called eruptive postoperative SCC can occur, in which cytokines released during wound healing triggering secondary tumor formation in genetically predisposed cells surrounding the original SCC. Given that lower extremity SCCs are less aggressive but more prone to surgical complications when excised, we believe they may be good candidates for localized non-surgical treatment.

5-fluorouracil (5FU) is a well-studied and characterized chemotherapeutic agent that has been used systemically, topically and intralesionally for a variety of malignancies and conditions including many dermatologic abnormalities.

It has been used on and off label topically for the treatment of actinic keratosis, SCC, superficial basal cell carcinoma, extra-mammary Paget's disease confined to the epidermis, Bowen's disease, porokeratosis, and genital warts. Intralesionally it has been used off label for the treatment of keloids, knuckle pads, warts, hypertrophic scars, basal cell carcinoma, and keratoanthoma.

Reports of its use intralesionally in invasive cutaneous SCC, other than in keratoacanthomas, are very limited. We are aware of 3 such reports in the literature. In the largest study to date, 6 weekly intralesional injections of 5FU-epinephrine gel were performed on 23 patients with cutaneous SCC on various body sites, 22 (96%) of whom demonstrated histologically confirmed tumor clearance. This study, however, used a proprietary gel formulation which is not widely available. There are two other case reports of successful treatment of SCC with 6-8 intralesional injections of 5FU at weekly intervals. The three published studies injected 0.6ml to 2.4ml of 5FU, per each weekly session, at concentrations of 30mg/ml to 50mg/ml.

Calcipotriene is a synthetic form of vitamin D. It is FDA approved for the treatment of psoriasis. Calcipotriene binds to vitamin D receptors on skin cells and helps regulate the growth and differentiation of skin cells. It inhibits keratinocyte proliferation (cell growth) and enhances keratinocyte differentiation. Recently it has been shown that calcipotriene acts as topical immune response modulator through induction of thymic stromal lymphoprotein (TSLP) expression. TSLP is an epithelium-derived cytokine and a regulator of allergic inflammation in the skin. It has been shown by Shadmehr Demehri et.al. that TSLP released by barrier-defective skin in mice blocks cancer development by recruiting T cells to mount robust antitumor immunity in the skin. The adaptive immune response mounted by TSLP against cancer can eliminate cancerous lesions in the skin and prevent new lesions from developing.

A recent clinical trial conducted by Demehri showed that participants treated with topical 5FU and topical and topical calcipotriene showed a significant reduction in the number of actinic keratosis (face, scalp, & upper extremities) vs topical 5FU and vaseline. Furthermore, the cohort treated with 5FU and topical and topical calcipotriene remained SCC-free for more than 1,500 days suggesting the induction of tissue-resident memory T (trm) cells.

The purpose of the study is to evaluate this relatively unexplored approach to the treatment of SCC on the lower extremities and establish a non-surgical therapy to improve outcomes, eliminate the need for surgery and mitigate the occurrence of infections and secondary tumor eruptions.

Topical 5FU is approved for the treatment of superficial basal cell carcinoma smaller than 2cm in diameter and not located on the feet, hands or feet but cannot penetrate deep enough into the skin to be an effective treatment for SCC. For the proposed phase I clinical trial, we propose to increase tumor accessibility by delivering 5FU directly and specifically to the tumor through intralesional injections to kill accessible localized SCC cells.

5FU is currently in clinical use with a well-established safety profile. It is anticipated that intralesional injections of 5FU will enable direct and specific delivery of chemotherapy to the tumor, thereby reducing the potential for systemic toxicity. Further, intralesional injections of-5FU enable tumoral delivery of locally effective concentrations of 5FU using doses that are orders of magnitude below those used currently for the intravenous (IV) treatment of multiple malignancies.

5FU alone may not be enough eliminate all the tumor cells so we also propose to treat a group of patients with intralesional 5FU and 0.005% calcipotriene topical cream. The goal is to develop a synergistic effect and establish an immune response to the 5FU induced apoptotic SCC cells in order to kill off any remaining SCC cells not undergoing apoptosis. Also 5FU alone may not be as effective of a treatment as the combination therapy in the prevention of secondary tumor recurrence. It is hoped that the combination therapy will induce a memory response and reduce the incidence of secondary tumors.

Participants will have at least 1 SCC lesion greater 1cm and less than 2 cm in largest diameter, on their lower extremities. The clinical diagnosis of SCC will be confirmed histologically by a deep shave biopsy of less than half of the lesion. The remainder of the lesion will be used for intralesional injections of 5FU or intralesional 5FU/topical calcipotriene according to the following schema:

In this study, a total of 30 patients will be randomly assigned into 3 groups. Randomization will be conducted using the UPCI randomizer, which is maintained by the Biostatistics Facility of UPCI (https://randomize.upci.pitt.edu/randomizer/home.seam). Group 1: 10 patients will serve as a control group, and will receive neither 5FU injection nor topical calcipotriene. Group 2: 10 patients, will receive a single intralesional injection of 50mg of 5FU in 1ml aqueous injectable solution once a month for 3 months. Group 3: 10 patients will receive intralesional 5FU as administered in the previous group. They will also apply a topical application of 0.005% calcipotriene cream to the same lesion two (2) times daily for four (4) days one (1) day after each of three (3) 5FU injections. At the time of initial consent, a 2 mm punch biopsy of the lesion will be obtained. Half of biopsy will be stored for tissue banking and future study, while the other half will be sent for histological confirmation of SCC. A second 2 mm punch biopsy will be taken at 2 months for mid-point analysis. This will be stored for tissue banking and future study. A month after the last injection (week 12), the lesion will be surgical resected in the non-treated Group 1 to render the participant's disease free. Resection is the current standard of care for these tumors. Participant's in Group 2 and 3 will have a 4mm punch biopsy will be taken to confirm pathologic resolution.

Depending on the results of the confirmational biopsy the following actions will be taken:

1. Negative biopsy - clinical follow-up every 6 months for 2 years
2. Positive biopsy - Mohs surgery 2-4 weeks after in In all groups part of, or all of, the resected tumor and surrounding skin will be stored for tissue banking and future study.

All lesions will be photographed and treatment response will be evaluated 4 weeks after the first 5FU injection prior to excision.

Inclusion Criteria:

• Biopsy-confirmed SCC more than 1.0 cm and less than 2.0 cm in diameter in the lower extremities, defined as the knees and below.
• Subjects must have an expected survival of greater than or equal to12 months.
• Subjects must not be on any other investigational device/drug treatment.
• Subjects must to be willing to adhere to the instructions of the Investigator and his research team and sign an Informed Consent Form prior to entry into the study.
• Patient is ≥ 18 years of age on day of signing informed consent.
• Patient must have a performance status of 0 or 1 on the Eastern Cooperative Oncology Group (ECOG) Performance Scale.
• Female patient of childbearing potential has a negative urine or serum pregnancy test within 7 days prior to receiving the first dose of study medication. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required. The serum pregnancy test must be negative for the patient to be eligible.
• Female patients enrolled in the study, who are not free from menses for > 2 years, post hysterectomy/oophorectomy, or surgically sterilized, must be willing to use either 2 adequate barrier methods or a barrier method plus a hormonal method of contraception to prevent pregnancy, or to abstain from heterosexual activity throughout the study, starting with the first dose of study drug at visit 1 through 120 days after the last dose of study drug. Approved contraceptive methods include for example: intra-uterine device, diaphragm with spermicide, cervical cap with spermicide, male condoms, or female condom with spermicide. Spermicides alone are not an acceptable method of contraception. Male patients must agree to use an adequate method of contraception starting with the first dose of study drug through 120 days after the last dose of study drug.

Exclusion Criteria:

• Patients with any evidence of nodal (Nx) and/or metastatic disease including distant subcutaneous and/or lymph node metastases.

• Patients with primary non-cutaneous SCC - such as nasopharyngeal SCC.

• Patient with history of receiving organ transplantation.

• Patients with history of iatrogenic systemic immunosuppression.

• Patients with a history of skin or other disorder(s),that in the opinion of the investigator, requires topical application of steroids and/or other creams/ointments.

• Patients with evidence of active infection - active and/or untreated hepatitis B/C, HIV, etc - requiring systemic therapy.

• Patients with a known history of autoimmune disease.

• Patients with the following cardiac co-morbidities including:

  • Baseline known prolongation of QT/QTc interval (QTc interval >500 msec).
  • Heart failure either on clinical examination (manifestations include ascites, cardiomegaly, dyspnea, edema, gallop rhythm, hepatomegaly, oliguria, pleural effusion, pulmonary edema, tachycardia) or based on known decreased left ventricular ejection fraction (LV EF) <50%.
  • Patients who have had chemotherapy, radioactive or biological cancer therapy within four weeks prior to the first dose of study drug, or who has not recovered to Common Terminology Criteria for Adverse Events (CTCAE) Grade 1 or better from the adverse effects (AEs) due to cancer therapeutics administered more than four weeks earlier. Subjects with ≤ grade 2 neuropathy are an exception to this criterion and may qualify for the study.
  • If patient received major surgery, they must have recovered adequately from the toxicity and/or complications from the intervention prior to starting therapy.

• Patients currently participating or who have participated in a study of an investigational agent or using an investigational device within 4 weeks of the first dose of study drug.

• Patients expected to require any other form of systemic or localized antineoplastic therapy while on study.

• Patients with a known history of a hematologic malignancy, primary brain tumor or sarcoma, or of another primary solid tumor, unless the patient has undergone potentially curative therapy with no evidence of that disease for five years.

  ° The time requirement also does not apply to patients who underwent successful definitive resection of basal or squamous cell carcinoma of the skin, superficial bladder cancer, in situ cancers including cervical cancer, breast cancer, melanoma, or other in situ cancers.

• Patients who have previously had a severe hypersensitivity reaction to 5-fluorouracil or imiquimod.

• Serious illnesses, such as: cardiovascular disease (uncontrolled congestive heart failure, hypertension, cardiac ischemia, myocardial infarction, and severe cardiac arrhythmia), bleeding disorders, autoimmune diseases, severe obstructive or restrictive pulmonary diseases, active systemic infections, and inflammatory bowel disorders.

• Patients with a history or current evidence of any condition, therapy, or laboratory abnormality that might confound the results of the study, interfere with the patient's participation for the full duration of the study, or are not in the best interest of the patient to participate, in the opinion of the treating Investigator.

• Patients who are, at the time of signing informed consent, regularly using illicit drugs or are recently (within the last year) abusing illicit substances (including alcohol).

• Patient is pregnant or breastfeeding, or expecting to conceive or father children within the projected duration of the study.

Individual participant data that underlie the results reported in this article, after deidentification (text, tables, figures, and appendices)

Immediately following publication. No end date.

Investigators whose proposed use of the data has been approved by an independent review committee (learned intermediary) identified for this purpose.

To achieve aims in the approved proposal.

Proposals may be submitted up to 36 months following article publication. Proposals should be directed to puglianomauroma@upmc.edu. To gain access, data requestors will need to sign a data access agreement. Data are available for 5 years through RedCap.