The purpose of the present trial was to determine the feasibility of the daily topical application of the piperidine nitroxide, MTS-01, combined with chemoradiotherapy in the treatment of patients with anal carcinoma. The secondary study endpoints were the description of the effects of this agent on skin toxicity and rectal-associated lymphoid tissue. The participants received radiotherapy concurrent with mitomycin-C and 5-fluorouracil for carcinoma of the anal canal. MTS-01 was applied to the bilateral inguinal area and the gluteal cleft. Dermatologic and non-dermatologic toxicity was graded throughout the treatment period. Circulating lymphocytes were serially collected for phenotyping. Rectal mucosal snag biopsies were collected at baseline and at 1 year of follow-up. A total of 5 patients received topical MTS-01. Adverse events attributed to MTS-01 included asymptomatic grade 1 hypoglycemia and grade 1–2 diarrhea. Dermatitis within untreated, radiated skin was not more severe than dermatitis in MTS-01-treated, unirradiated skin. Circulating CD4+ lymphocyte suppression was noted at >1 year following treatment in human immunodeficiency virus-negative participants. CD4+ lymphocytes remained suppressed in the irradiated rectal mucosa at 1 year, whereas the CD8+ lymphocyte numbers recovered or increased. On the whole, the present study demonstrates that the MTS-01 topical application was tolerable with minimal toxicity. Chemoradiation for anal cancer led to prolonged CD4+ lymphocytopenia in the circulation and gut mucosa.
There are ~8,300 cases and 1,280 related deaths due to carcinoma of the anal canal each year in the United Sates (
Radiation dermatitis is one of the most common severe toxicities observed during chemoradiation for anal cancer. In Radiation Therapy Oncology Group (RTOG) 0529 (
MTS-01 (Tempo1; 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a nitroxide oxygen radical scavenger that has been formulated as a topical gel (tempol 70 mg/ml in water, ethanol and hydroxylpropyl cellulose). The nitroxides are a class of stable free radical compounds that exhibit antioxidant activity, protecting mammalian cells against hydrogen peroxide, superoxide and t-butyl hydroperoxide cytotoxicity (
The primary objective of the present study was to assess the safety and tolerability of delivering a topical Tempol application on a daily basis prior to irradiation in the inguinal area and gluteal cleft of patients receiving combined therapy with MMC, 5-FU and radiation therapy for carcinoma of the anal canal. The secondary objectives included the description of the severity of skin toxicity with this regimen and the need for treatment breaks.
Patients with histologically proven invasive primary squamous carcinoma of the anal canal, stage T1-4, N0-3, M0, with no previous therapy for anal cancer were eligible for this National Cancer Institute Institutional Review Board-approved clinical trial (NCT01324141; registered on March 28, 2011). All research was performed in accordance with relevant guidelines and regulations. All studies reported were outlined in an informed consent document signed by all participants. The study subjects were >18 years of age with an Eastern Cooperative Oncology Group (ECOG) performance status ≤2 and adequate bone marrow, renal and hepatic functions. Patients with human immunodeficiency virus (HIV) and a CD4 T-cell count >100 cells/µl and an ECOG performance status <2 were eligible.
Participants were simulated in the supine position at 1 h following oral contrast administration with a marker placed at the anal verge. CT images were obtained through the pelvis and inguinal regions. Contouring or targets and critical structures was performed using Eclipse software (v4, Varian Medical Systems, Inc.) and based on the RTOG Consensus guidelines for rectal and anal cancer planning (
MMC was delivered intravenously at a dose of 10 mg/m2 (maximum 20 mg) on days 1 and 29. 5-FU was delivered at the dose of 1,000 mg/m2/day as a 96-h continuous venous infusion on days 1 and 29. Radiotherapy commenced concurrently with chemotherapy (day 1) using IMRT.
To guide the MTS-01 application, an anterior projection of the body surface with the prescription isodose volumes [primary planning target volume (PTV) and nodal PTV] was generated using Eclipse software v4 (
Adverse events (AE) were assessed throughout treatment and until 4 weeks of follow-up using the Common Terminology Criteria for Adverse Events (CTCAE v4.0,
Optional rectal mucosal snag biopsies were obtained during flexible sigmoidoscopy performed at baseline and at 12 months following the completion of treatment, and processed to a single cell suspension as previously described (
Aliquots of plasma were collected prior to treatment (baseline) and course 1 (day 28) and stored at −80°C until use. The concentrations of interleukin (IL)-7, transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor A (VEGF-A) in plasma were determined using Meso Scale Discovery multiplex chemiluminescent assays as per the manufacturer's recommended protocol, and analyzed using a S6000 Instrument (Meso Scale Diagnostics LLC).
A total of 5 patients were enrolled in the study. All participants completed chemoradiation and MTS-01 treatment. The patient demographics are summarized in
AEs attributed to MTS-01 were rare, with the majority of AEs being attributed to chemotherapy or radiation. There were no dose-limiting toxicities. In all cases, toxicities possibly attributed to MTS-01 were also possible toxicities of chemoradiotherapy or concurrent medications. For example, the only grade 3 toxicities possibly attributable to MTS-01 were a decrease in the CD4+ T cell count in a single patient, and a single brief episode of grade 3 fatigue, which were also attributable to chemoradiotherapy. Another patient experienced grade 2 diarrhea, and all remaining toxicities were grade 1, including fatigue and hypoglycemia (
There were several grade 3 or higher AEs attributed to either IMRT, MMC or 5-FU treatment that are summarized in
As this trial aimed to assess gut-associated lymphoid tissue as an exploratory endpoint, the serial lymphocyte phenotyping of blood was performed as a comparator for tissue studies throughout treatment and follow-up. As expected with chemoradiotherapy, leukopenia was pronounced soon following treatment initiation (
In total, 1 patient (HIV-positive, stage T4 disease) developed rapid disease progression outside of the radiation field following treatment and was removed from the study. The remaining 4 patients are alive and relapse-free with no evidence of disease throughout the duration of follow-up.
All 5 patients experienced radiation dermatitis in the radiation treatment field. Radiation dermatitis within the MTS-01-treated areas and the control areas was assessed at each time point during examination by the treating physician (
Toxicity in the MTS-01-treated gluteal cleft was more severe than that in other assessed sites (
To describe global pain during treatment, the brief pain inventory was administered weekly during treatment and in the subsequent follow-up period. As demonstrated in
Rectal mucosal biopsies were obtained from 3 consenting patients at baseline and at 1 year following the completion of treatment. The analysis of lymphocyte subsets in these biopsy tissues revealed a reduction in CD3+ and CD4+ T-cells in all patients at the 1-year follow-up (
As aforementioned, leukopenia and lymphopenia were rapid and often profound with the chemoradiotherapy delivered in this trial. The evaluation of cytokines known to play a role in lymphopoiesis were analyzed at baseline vs. the end of the first course of chemotherapy (course 1, day 28). In 4 of the 5 participants, the IL-7 levels increased at course 1 (day 28) relative to baseline levels, whereas the TGF-β1 concentrations in the circulation decreased universally. No clear patterns were observed in the plasma concentrations of TNF-α and VEGF (
The primary objective of the present study was to assess the safety and efficacy of delivering topical MTS-01 daily prior to irradiation in the bilateral inguinal area and gluteal cleft of patients receiving combined therapy with MMC, 5-FU and radiation therapy for carcinoma of the anal canal. In the present phase I study on 5 patients, minimal toxicity was noted with the application of MTS-01. A strong signal of efficacy was not demonstrated, although there are significant limitations to this observation in this small-scale study. The present study reported a long-term decrease in leukocyte counts, specifically CD4+ lymphocytes, associated with standard chemoradiation for anal carcinoma, with evidence of CD8+ lymphocyte persistence or recovery in tissue relative to circulation and relative to CD4+ lymphocytes. Chemoradiotherapy combined with MTS-01 led to a universal decrease in TGF-β1 levels.
The present study employed several methods to increase the likelihood of determining the efficacy of radioprotection. Dermatitis was scored in real-time by a single trained physician using control sites in each patient. A blinded observer assessed response using deidentified professionally captured medical photographs of the sites scored for toxicity. These images were collected in identical locations, with identical lighting conditions and identical photography equipment.
However, there were also several limitations to the ability to demonstrate the efficacy of MTS-01 as a radioprotector of skin in the present study. An inherent challenge in the evaluation of topical radioprotectors is the difficulty of predicting locations of severe dermatitis in an individual patient. The inguinal control site (no MTS-01 applied) was situated in the center of the inguinal region, below the inguinal fold, and was specifically selected to reduce the chance of MTS-01 contamination of the site during hip flexion when patients rose to walk to radiation treatment following the MTS-01 application. Although the skin surrounding this control site, where MTS-01 was not applied, often had less dermatitis than the more medial portions of the inguinal region, toxicity was scored based on the most severe toxicity within the inguinal region, which may have resulted in toxicity grading spuriously appearing to reflect less toxicity at the inguinal control site (MTS-01 not applied, radiated) relative to the remaining inguinal areas. Thus, even if zones of toxicity can be accurately predicted, control sites must be carefully selected to ensure accurate comparisons of efficacy, the simultaneous goals of both minimizing contamination and ensuring toxicity grading accounts for regional variation in dermatitis. The inclusion of only 5 patients prior to study closure also prevented firm conclusions regarding the efficacy of MTS-01. Regardless, the lessons learned from the techniques utilized in the present study may be useful in designing future studies assessing topical radioprotectors or mitigators.
Despite an inability to demonstrate a reduction in dermatitis with MTS-01, there was minimal toxicity to its application, even in the setting of evolving dermatitis. Consistent with the findings of other clinical trials in cancer patients receiving topical formulations of Tempol, the most commonly reported AEs were gastrointestinal, constitutional, dermatological and metabolic (
The lack of systemic absorption of MTS-01 in previous research (
Although hematologic toxicity is frequently described as a consequence of chemoradiation for carcinoma of the anal canal, a strength of the present study was a more comprehensive evaluation of lymphocytopenia in a small patient subset. All participants underwent the serial assessment of blood counts and lymphocyte phenotyping. In addition, the four HIV seronegative participants without progression who were follow-up for 1 year following the completion of therapy were noted to have prolonged lymphocytopenia. Lymphocytopenia was largely due to prolonged decreases in the numbers of CD4+ T-cells, while circulating CD8+ lymphocytes recovered in the majority of patients to a normal range within weeks following treatment. These finding were true even in the setting of increases in the levels of the homeostatic cytokine, IL-7, suggesting that physiological responses to lymphocytopenia may be inadequate to promote CD4 reconstitution over a period of 1 year after standard chemoradiation for anal cancer.
Previous research has demonstrated the suppression of CD4+ lymphocytes in HIV-positive individuals following chemoradiotherapy for anal cancer (
A notable component of the present study is the assessment of CD4+ and CD8+ lymphocytes in rectal mucosal biopsies at 1 year following irradiation compared to baseline levels. Although both circulating CD4 and CD8 cells remained suppressed at 1 year, only the numbers of CD4+ lymphocytes were reduced in rectal tissue at 1 year. Preclinical studies and limited human tissue studies suggest that although T-cells do not account for the majority of accumulated cells in irradiated tissue (
The design of the present study does not allow for the ruling out of the possibility that these profound and sustained immunosuppressive effects were related to MTS-01 administration. However, other clinical studies evaluating MTS-01 have not identified prolonged immune effects, and the available literature that has described the lymphocyte count and CD4 count suppression when combining chemotherapy and radiotherapy for the treatment of other cancers further supports that the causative agents are chemotherapy and radiation (
Another observation was that treatment with chemoradiation combined with MTS-01 led to a decrease in plasma levels of TGF-β1. TGF-β1 has been implicated in the pathogenesis of human papillomavirus-associated malignancies (
In conclusion, as demonstrated herein, MTS-01 is tolerable when used to manage dermatotoxicity in patients with localized anal cancer undergoing chemoradiation. The lack of an efficacy signal that was noted to be due to the inadequate sample size and control site selection, were significant factors in the decision to close the study to accrual early. A more suitable control site that would not be subject to easy cross-contamination with MTS-01, but would be expected to develop severe dermatitis, could not be identified. Regardless, there are important lessons to be learnt for future studies evaluating a topical radiation protector and attempting to integrate a control site. Despite the sample size, there are several interesting hypotheses generating findings related to treatment induced CD4 lymphocytopenia and TGF-β1 that provide subjects for future studies.
The authors are grateful to Ms. Luz Giordano and Ms. Debbie McNally, both from the National Cancer Institute, for their contributions to the conduct of this trial. Both were involved in the application of the investigational agent and the conduct of research-related assessments. The trial registration number for the study is NCT01324141 (registered on March 28, 2011).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
DC and TU were involved in the conception and design of the study, in the acquisition of data, data analysis, interpretation of the data and in manuscript preparation. LV was involved in data analysis, interpretation of the data and in manuscript preparation. KC was involved the acquisition of data, data analysis and in manuscript preparation. TCZ, DS and MY were involved in the acquisition of data and in manuscript preparation. JBM was involved in the conception of the study and in manuscript preparation. WT was involved in data analysis and in manuscript preparation. IS was involved in the acquisition of data, data analysis, interpretation of the data and in manuscript preparation. DC and KC confirm the authenticity of the raw data. All authors have read and approved the final manuscript.
All analyses reported in the present study relating to human subjects were reviewed and approved by the National Cancer Institute Institutional Review Board. All studies reported were outlined in an informed consent document signed by all participants.
Not applicable.
The authors declare that they have no competing interests.
MTS-01 application site. A surface rendering of the patient's body was generated in the Eclipse treatment planning system. The planning target volume for the pelvic lymph nodes (blue) and the primary tumor planning target volume (red) were overlaid and the dose to the skin was reviewed. An MTS-01 treatment area was selected based on these volumes and the dose received by the skin in this area. A 2×2 cm control site was selected in the inguinal area (Control 1) where no MTS-01 was applied. A second control site was selected near the umbilicus outside of the radiation field where MTS-01 was applied was also selected (Control 2). MTS-01 was applied to the areas outlined in white.
Circulating leukocyte and lymphocyte counts during and after treatment. Complete blood counts and lymphocyte phenotyping by a clinical laboratory was conducted weekly during chemoradiation and then at varying intervals throughout the duration of follow-up. Individual patients are color-coded consistently across the graphs for (A) total white blood cell count, (B) absolute lymphocyte count, (C) CD4+ lymphocyte count, and (D) CD8+ lymphocyte count. The lower limit of normal (based on the clinical laboratory that conducted the assay) for each measure is noted as a hashed line. CTCAE v4.0 toxicity grades are noted, with the exception of CD8+ lymphocytes, where CTCAE toxicity was not defined. The data of the 1 patient who was positive for human immunodeficiency virus are represented by the green-colored line. CTCAE, Common Terminology Criteria for Adverse Events.
RTOG Skin Toxicity Grading. RTOG acute skin toxicity in MTS-01 treatment and control areas was graded at baseline and weekly during treatment, and then at weeks 1, 2 and 4 of follow-up. Each site was scored separately by (A) a single treating physician at the point of care or (B) by a blinded radiation oncologist via review of professionally acquired medical photographs and presented as a mean of scores for all participants at each time point. Maximum grade of RTOG skin toxicity (C) and time to highest grade acute skin toxicity (D) are graphed for each site as a mean of all patients with standard deviation. RTOG, Radiation Therapy Oncology Group.
Circulating and peripheral lymphocyte subsets. Biopsy tissue from the colon and blood was collected at baseline and at 1 year of follow-up. Tissue was dissociated to individual cells, fixed and subjected to flow cytometric analyses for cell surface markers (representative flow cytometry plots for these data are available from the corresponding author on reasonable request). All patients who consented to the tissue biopsy were HIV-negative. Colors correspond to individual patients, and those reported in
Patient demographics.
Characteristic | No. of patients (%) |
---|---|
Sex | |
Male | 2 (40) |
Female | 3 (60) |
Age, years | |
Range | 49-63 |
Median | 57 |
Race | |
African-American | 1 (20) |
Caucasian | 4 (80) |
ECOG status | |
0 | 3 (60) |
1 | 2 (40) |
2 | 0 (0) |
3 | 0 (0) |
4 | 0 (0) |
5 | 0 (0) |
HIV status | |
Positive | 1 (20) |
Negative | 4 (80) |
HPV status (anal swab) | |
Positive | 0 (0) |
Negative | 5 (100) |
T stage (AJCC 7th edition) | |
T1 | 0 (0) |
T2 | 2 (40) |
T3 | 3 (60) |
T4 | 0 (0) |
N stage (AJCC 7th edition) | |
N0 | 4 (80) |
N1 | 0 (0) |
N2 | 0 (0) |
N3 | 1 (20) |
Disease stage (AJCC 7th edition) | |
I | 0 (0) |
II | 4 (80) |
IIIA | 0 (0) |
IIIB | 1 (20) |
IV | 0 (0) |
AJCC, American Joint Committee on Cancer (
Adverse events observed in the present study trial.
Type of adverse event | MTS-01 Grade 1 | MTS-01 grade 2 | MTS-01 Grade 3 | 5-FU/MMC/IMRT Grade 2 | 5-FU/MMC/IMRT Grades 3–4 |
---|---|---|---|---|---|
Non-hematologic | |||||
Radiation dermatitis | 2 | 3 | |||
Nausea | 3 | ||||
Vomiting | 2 | ||||
Abdominal pain | 1 | ||||
Diarrhea | 1 | 1 | 1 | 2 | |
Gastroesophageal reflux | 1 | ||||
Mucositis | 1 | ||||
Urinary tract pain | 2 | ||||
Bladder spasm | 1 | ||||
Urinary incontinence | 1 | ||||
Fatigue | 1 | 1 | 1 | ||
Hypoglycemia | 1 | ||||
Transaminitis | 1 | ||||
Hypoalbuminemia | 1 | ||||
Hypocalcemia | 1 | ||||
Myalgia | 1 | ||||
Headache | 1 | ||||
Syncope | 1 | ||||
Infection | 1 | 1 | |||
Insomnia | 1 | ||||
Pain | 2 | 2 | |||
Hematologic | |||||
Leukopenia | 5 | ||||
Lymphopenia | 5 | ||||
Neutropenia | 2 | 3 | |||
Febrile neutropenia | 1 | ||||
Thrombocytopenia | 2 | ||||
Anemia | 3 | ||||
CD4 count decrease | 1 |
5-FU, 5-fluorouracil; MMC, mitomycin-C; IMRT, intensity modulated radiation therapy.
Brief pain inventory scores.
Baseline | Treatment week 3 | Treatment week 6 | 1-Month follow-up | 3-Month follow-up | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Pain inventory | Mean | Range | Mean | Range | Mean | Range | Mean | Range | Mean | Range |
Worst pain | 4.8 | 0-10 | 3.5 | 0-8 | 7.2 | 4-10 | 3.1 | 0-9 | 2.1 | 0-5 |
Least pain | 1.4 | 0-4 | 1.5 | 0-4 | 3.6 | 1-5 | 1.2 | 0-3 | 2.8 | 0-5 |
Average pain | 1.8 | 0-4 | 1.9 | 0-3.5 | 5.0 | 2-7 | 2.0 | 0-6 | 3.0 | 0-6 |
Pain at time of survey | 1.6 | 0-6 | 1.3 | 0-5 | 5.4 | 2-9 | 2.2 | 0-6 | 2.2 | 0-5 |
% Pain relief after medication | 90.0 | 60-100 | 93.8 | 85-100 | 44.0 | 10-90 | 89.0 | 75-100 | 83.0 | 50-100 |
Pain interference | 1.73 | 0.33-3.89 | 3.00 | 0.44-8.00 | 6.70 | 2.89-9.44 | 3.17 | 0-8.33 | 1.87 | 0-4.56 |