Executive Summary of the American Radium Society® (ARS) Appropriate Use Criteria (AUC) for Non-Operative Management (NOM) for Rectal Adenocarcinoma: Systematic Review and Guidelines.

PURPOSE: For rectal cancer patients, the standard approach of chemotherapy, radiation therapy (RT), and surgery (Trimodality Therapy, TMT) is associated with significant long-term toxicity and/or colostomy for most patients. Patient options focused on quality-of-life (QOL) have dramatically improved, but there remains limited guidance regarding comparative effectiveness. This systematic review and associated guidelines evaluate how various treatment strategies compare to each other in terms of oncologic outcomes and QOL. MATERIALS AND METHODS: Cochrane and PRISMA methodology were used to search for prospective and retrospective trials and meta-analyses of adequate quality within the Ovid Medline database between 1/1/2012-6/15/2023. These studies informed the expert panel, which rated the appropriateness of various treatments in 6 clinical scenarios through a well-established consensus methodology (modified Delphi). RESULTS: The search process yielded 197 articles that advised voting. Increasing data show non-operative management (NOM) and primary surgery result in QOL benefits noted over TMT without detriment to oncologic outcomes. For rectal cancer patients for whom TME would result in permanent colostomy or inadequate bowel continence, NOM was strongly recommended as usually appropriate. Restaging with tumor response assessment 8-12 weeks following completion of RT/CRT was deemed a necessary component of NOM. The panel recommended active surveillance in the setting of a near complete or complete response. In the setting of NOM, 54-56 Gy in 27-33 fractions concurrent with chemotherapy and followed by consolidation chemotherapy was recommended. The panel strongly recommends primary surgery as usually appropriate for a T3N0 high rectal tumor for whom LAR and adequate bowel function is possible, with adjuvant chemotherapy considered if N+. CONCLUSIONS: Recent data supports NOM and primary surgery as important options that should be offered to eligible patients. Considering the complexity of multi-disciplinary management, patients should be discussed in a multi-disciplinary setting and therapy should be tailored to individual patient goals/values.

Oncologic Outcomes of Salvage Abdominoperineal Resection for Anal Squamous Cell Carcinoma Initially Managed with Chemoradiation.

Background: Abdominoperineal resection (APR) has been advocated for persistent or recurrent disease after failure of chemoradiation (CRT) for anal squamous cell cancer (SCC). Treatment with salvage APR can potentially achieve a cure. This study aimed to analyze oncological outcomes for salvage APR in a recent time period at a comprehensive cancer center. Methods: A retrospective review of all patients who underwent APR for biopsy-proven persistent or recurrent anal SCC between 1 January 2007 and 31 December 2020 was performed. Patients with stage IV disease at the time of initial diagnosis and patients with missing data were excluded. Univariate analysis was used with a chi-square test for categorical variables, and non-parametric tests were used for continuous variables. Kaplan-Meier survival analysis was performed to evaluate disease-specific (DSS), post-APR local recurrence-free (RFS), and disease-free survival (DFS). Results: A total of 96 patients were included in the analysis: 39 (41%) with persistent disease and 57 (59%) with recurrent SCC after chemoradiation had been completed. The median follow-up was 22 months (IQR 11-47). Forty-nine patients (51%) underwent extended APR and/or pelvic exenteration. Eight (8%) patients developed local recurrence, 30 (31%) developed local and distant recurrences, and 16 (17%) developed distant recurrences alone. The 3-year DSS, post-APR local recurrence-free survival, and disease-free survival were 53.8% (95% CI 43.5-66.5%), 54.5% (95% CI 44.4-66.8%), and 26.8% (95% CI 18.6-38.7%), respectively. In multivariate logistic regression analysis, positive microscopic margin (OR 10.0, 95% CI 2.16-46.12, p = 0.003), positive nodes in the surgical specimen (OR 9.19, 95% CI 1.99-42.52, p = 0.005), and lymphovascular invasion (OR 2.61 95% CI 1.05-6.51, p = 0.04) were associated with recurrence of disease. Gender, indication for APR (recurrent vs. persistent disease), HIV status, extent of surgery, or type of reconstruction did not influence survival outcomes. Twenty patients had targeted tumor-sequencing data available. Nine patients had PIK3CA mutations, seven of whom experienced a recurrence. Conclusions: Salvage APR for anal SCC after failed CRT was associated with poor disease-specific survival and low recurrence-free survival. Anal SCC patients undergoing salvage APR should be counseled that microscopic positive margins, positive lymph nodes, or the presence of lymphovascular invasion in the APR specimen are prognosticators for disease relapse. Our results accentuate the necessity for additional treatment strategies for the ongoing treatment challenge of persistent or recurrent anal SCC after failed CRT.

Radiation Therapy Physics Quality Assurance and Management Practices in Low- and Middle-Income Countries: An Initial Pilot Survey in Six Countries and Validation Through a Site Visit.

Purpose: Our purpose was to assess physics quality assurance (QA) practices in less resourced radiation therapy (RT) centers to improve quality of care. Methods and Materials: A preliminary study was conducted in 2020 of 13 select RT centers in 6 countries, and in 2021, our team conducted onsite visits to all the RT centers in Ghana, one of the countries from the initial survey. The RT centers included 1 private and 2 public institutions (denoted as Public-1 and Public-2). Follow-up surveys were sent to 17 medical physicists from the site visit. Questions centered on the topics of equipment, institutional practice, physics quality assurance, management, and safety practices. Qualitative and descriptive methods were used for data analysis. Questions regarding operational challenges (machine downtime, patient-related issues, power outages, and staffing) were asked on a 5-point Likert scale. Results: The preliminary survey from 2020 had a 92% response rate. One key result showed that for RT centers in lower gross national income per capita countries there was a direct correlation between QA needs and the gross national income per capita of the country. The needs identified included film/array detectors, independent dose calculation software, calibration of ion chambers, diodes, thermoluminiscence diodes (TLDs), phantoms for verification, Treatment Planning System (TPS) test phantoms, imaging test phantoms and film dosimeters, education, and training. For the post survey after the site visit in 2021, we received a 100% response rate. The private and the Public-1 institutions each have computed tomography simulators located in their RT center. The average daily patient external beam workload for each clinic on a linear accelerator was: private = 25, Public-1 = 55, Public-2 = 40. The Co-60 workload was: Public-1 = 45, Public-2 = 25 (there was no Co-60 at the private hospital). Public-1 and -2 lacked the equipment necessary to conform to best practices in Task Group reports (TG) 142 and 198. Public-2 reported significant operational challenges. Notably, Public-1 and -2 have peer review chart rounds, which are attended by clinical oncologists, medical physicists, physicians, and physics trainees. All 17 physicists who responded to the post site visit survey indicated they had a system of documenting, tracking, and trending patient-related safety incidents, but only 1 physicist reported using International Atomic Energy Agency Safety in Radiation Oncology. Conclusions: The preliminary study showed a direct correlation between QA needs and the development index of a country, and the follow-up survey examines operational and physics QA practices in the RT clinics in Ghana, one of the initial countries surveyed. This will form the basis of a planned continent-wide survey in Africa intended to spotlight QA practices in low- and middle-income countries, the challenges faced, and lessons learned to help understand the gaps and needs to support local physics QA and management programs. Audits during the site visit show education and training remain the most important needs in operating successful QA programs.

Radiation Oncology Residency Training Program Integration of Diversity, Equity, and Inclusion: An Association of Residents in Radiation Oncology Equity and Inclusion Subcommittee Inaugural Program Director Survey.

PURPOSE: The aim of this study was to investigate United States (US) radiation oncology (RO) program directors' (PDs) attitudes and practices regarding racial/ethnic diversity, equity, and inclusion (DEI) to better understand potential effects on underrepresented in medicine (UIM) residents in RO. METHODS AND MATERIALS: A 28-item survey was developed using the validated Ethnic Harassment Experiences Scale and the Daily Life Experiences subscale, as well as input from DEI leaders in RO. The survey was institutional review board-approved and administered to RO PDs. PDs were provided with the American Association of Medical Colleges definition of UIM, that is, "Underrepresented in medicine means those racial and ethnic populations that are underrepresented in the medical profession relative to their numbers in the general population." Descriptive statistics were used in analysis. RESULTS: The response rate was 71% (64/90). Institutional Culture and Beliefs: 42% responded that they had a department DEI director. A minority (17%, n = 11) agreed "I believe that people from UIM backgrounds have equal access to quality tertiary education in the US." The majority (97%, n = 62) agreed "My program values residents from UIM backgrounds." Support and Resources: The majority (78%, n = 50) agreed "My program has resources in place to assist/provide support for resident physicians from UIM backgrounds." Interview and Recruitment: Most PDs (53%) had not taken part in activities aimed at recruiting UIM residents and 17% had interviewed no UIM applicants in the past 5 years for residency. Resident Experiences of Racism: 17% (n = 11) agreed "UIM residents in my program have reported incidents of racism to me," and 28% (n = 18) agreed "I believe that UIM residents in my program have been treated differently because of their race/ethnicity by faculty, staff, coresidents or patients." CONCLUSIONS: Most PDs reported that they did not believe that UIM residents were treated differently in their department because of their race/ethnicity, and only a minority had received reports of racial discrimination experienced by residents. These data contrast resident experiences and suggest a disconnect between DEI perceptions and resident experiences among US RO PDs that should be addressed through increased programmatic action and evaluation.

Tailored Mentorship for the Underrepresented and Allies in Radiation Oncology: The Association of Residents in Radiation Oncology Equity and Inclusion Subcommittee Mentorship Experience.

PURPOSE: There are limited opportunities for mentorship for underrepresented in medicine (URM) trainees and physicians in radiation oncology (RO). The purpose of this study was to create and evaluate a formal mentorship program open to URMs and allies with interests in diversity, equity, and inclusion. METHODS AND MATERIALS: A mentorship program incorporating a virtual platform was designed by the Association of Residents in Radiation Oncology Equity and Inclusion Subcommittee. It was structured to include 6 sessions over 6 months with matched mentor-mentee pairs based on responses to a publicized online interest form. A compilation of evidence-based guidelines was provided to optimize the mentorship relationship. Linked pre- and postprogram surveys were administered to collect demographic data, define baseline goals and level of support, and evaluate program satisfaction. RESULTS: Thirty-five mentor-mentee pairs were matched; 31 mentees completed the preprogram survey and 17 completed the postprogram survey. Preprogram, only 3 mentees (9.7%) reported satisfaction with current mentorship and 5 (16%) reported mechanisms or mentorship in place at their program to support URMs. On the postprogram survey, mentees reported high satisfaction with areas of mentorship, mentor attributes, and the program overall. Opportunities for improvement include implementation of mechanisms to enhance communication with mentor-mentee pairs and maintain longitudinal engagement. CONCLUSIONS: In the first tailored mentorship program in RO for URMs and those with diversity, equity, and inclusion interests, our results demonstrate that there is self-reported interest for better mentorship for URMs in RO, and that a nationwide structured mentorship program can address participants' goals with high satisfaction. Program expansion could provide URMs and allies in RO more opportunities for career development and promote a greater sense of community and inclusion within the field.

Radiation Oncology Resident Perspectives on Diversity, Equity, and Inclusion: An Association of Residents in Radiation Oncology Equity and Inclusion Subcommittee Inaugural Resident Survey.

PURPOSE: In this study, radiation oncology residents were surveyed on perceptions of diversity, equity, inclusion, and belonging in their residency training programs. METHODS AND MATERIALS: A 23-item survey was developed by the Association of Residents in Radiation Oncology Equity and Inclusion Subcommittee resident members and faculty advisors. The survey was divided into 4 sections: institutional culture, support and resources, interview and recruitment, and experiences of bias. The survey was sent individually to residents from all Accreditation Council for Graduate Medical Education-accredited radiation oncology programs. RESULTS: The survey was issued to 757 residents. A total of 319 residents completed the survey, for a response rate of 42%. All postgraduate years and geographic regions were represented. Significant racial, ethnic, and gender differences were present in survey response patterns. White residents (94%, 164 of 174) and male residents (96%, 186 of 194) were more likely to strongly agree/agree that they were treated with respect by their colleagues and their coworkers than other racial groups (P < .005) or gender groups (P < .008). Only 3% (5 of 174) of White residents strongly agreed/agreed that they were treated unfairly because of their race/ethnicity, while 31% (5 of 16) of Black residents and 10% (9 of 94) of Asian residents strongly agreed/agreed (P < .0001). Similarly, Hispanic residents were more likely to strongly agree/agree (24%, 5 of 21) than non-Hispanic residents (7%, 20 of 298) (P = .003). Regarding mentorship, there were no differences by gender or ethnicity. There were differences by race in residents reporting that they had a supportive mentor (P = .022), with 89% (154 of 174) of White residents who strongly agreed/agreed, 88% (14 of 16) of Black residents, and 91% of Asian residents (86 of 94). CONCLUSIONS: This survey reveals that experiences of support, mentorship, inclusion, and bias vary significantly among radiation oncology residents based on race, ethnicity, and gender. Radiation oncology has opportunity for growth to ensure an equitable experience for all residents.

American Radium Society (ARS) Appropriate Use Criteria (AUC) for Extrahepatic Cholangiocarcinoma.

Although uncommon, extrahepatic cholangiocarcinoma (EHCC) is a deadly malignancy, and the treatment approaches remain controversial. While surgery remains the only cure, few patients are candidates for resection up front, and there are high rates of both local and distant failure following resection. Herein, we systematically review the available evidence regarding treatment approaches for patients with EHCC, including surgery, radiation, and chemotherapy. The evidence regarding treatment outcomes was assessed using the Population, Intervention, Comparator, Outcome, and Study design (PICOS) framework. A summary of recommendations based on the available literature is outlined for specific clinical scenarios encountered by providers in the clinic to guide the management of these patients.

Patterns of Care and Utilization Disparities in Proton Radiation Therapy for Pediatric Central Nervous System Malignancies.

Purpose: Proton radiation therapy (PR) is well established in the treatment of pediatric malignancies in the central nervous system (CNS) with dosimetric advantages that reduce late radiation therapy (RT) effects. In this analysis, we sought to evaluate the utilization of PR in children with primary CNS malignancies and characterize the clinical and sociodemographic factors predictive of receipt of PR. Methods and Materials: The National Cancer Database was queried to identify all pediatric patients with primary CNS malignancies treated with curative intent RT from 2004 to 2017. Clinical characteristics and demographics were analyzed using standard t and χ2 testing. Predictors of PR receipt were identified with univariable and multivariable logistic regression. Results: We identified 9126 patients ≤18 years of age treated with RT between 2004 and 2017, of which 1045 (11.5%) received PR. PR usage continued to increase significantly, from <1% in 2004 to 28% in 2017. The proportion of white and Asian patients receiving PR for nonhigh-grade glioma and nonmeningioma CNS malignancies during the study period rose from <1% for both to 35% and 44%, respectively, and in black patients the proportion rose from <1% to 26%. Multivariable predictors of receipt of PR include year of diagnosis, age <6 years, income level, distance from PR facility, and histology; multivariable predictors of receipt of photon RT include black race, rural residence, and Medicaid insurance. These factors remained significant when isolating the most recent 5 years of data. Conclusions: Proton radiation therapy usage for CNS malignancies increased significantly during the study period. Despite the potential clinical advantages of PR for pediatric primary CNS malignancies, there are notable socioeconomic, geographic, and racial disparities in the receipt of PR that persisted despite the increased availability and accessibility. Further study is warranted to identify how to address the disparities and better support these patients.

Intensity-Modulated Proton Therapy for Nasopharynx Cancer: 2-year Outcomes from a Single Institution.

PURPOSE: Advances in radiotherapy have improved tumor control and reduced toxicity in the management of nasopharyngeal carcinoma (NPC). Local failure remains a problem for some patients with advanced primary tumors, and toxicities are significant given the large treatment volume and tumor proximity to critical structures, even with modern photon-based radiotherapy. Proton therapy has unique dosimetric advantages, and recent technological advances now allow delivery of intensity-modulated proton therapy (IMPT), which can potentially improve the therapeutic ratio in NPC. We report our 2-year clinical outcomes with IMPT for NPC. MATERIALS AND METHODS: We retrospectively reviewed treatment records of patients with NPC treated with IMPT at our center. Demographics, dosimetry, tumor response, local regional control (LRC), distant metastasis, overall survival, and acute and late toxicity outcomes were reviewed. Analyses were performed with descriptive statistics and Kaplan-Meier method. Toxicity was graded per Common Terminology Criteria for Adverse Events (version 4.0). RESULTS: Twenty-six patients were treated from 2015 to 2020. Median age was 48 years (range, 19-73 years), 62% (n = 16) had T3-T4 disease, 92% (n = 24) were node positive, 92% (n = 24) had stage III-IV disease, and 69% (n = 18) had positive results for Epstein-Barr virus. Dose-painted pencil-beam IMPT was used. Most patients (85%; 22 of 26) were treated with 70 Gy(RBE) in 33 fractions once daily; 4 (15%) underwent hyperfractionated accelerated treatment twice daily. All received concurrent cisplatin chemotherapy; 7 (27%) also received induction chemotherapy. All patients (100%) completed the planned radiotherapy, and no acute or late grade 4 or 5 toxicities were observed. At median follow-up of 25 months (range, 4-60), there were 2 local regional failures (8%) and 3 distant metastases (12%). The Kaplan-Meier 2-year LRC, freedom from distant metastasis, and overall survival were 92%, 87%, and 85% respectively. CONCLUSION: IMPT is feasible in locally advanced NPC with early outcomes demonstrating excellent LRC and favorable toxicity profile. Our data add to the growing body of evidence supporting the clinical use of IMPT for NPC.

The Case for Brachytherapy: Why It Deserves a Renaissance.

The recent global events related to the coronavirus disease of 2019 pandemic have significantly changed the medical landscape and led to a shift in oncologic treatment perspectives. There is a renewed focus on preserving treatment outcomes while maintaining medical accessibility and decreasing medical resource utilization. Brachytherapy, which is a vital part of the treatment course of many cancers (particularly prostate and gynecologic cancers), has the ability to deliver hypofractionated radiation and thus shorten treatment time. Studies in the early 2000s demonstrated a decline in brachytherapy usage despite data showing equivalent or even superior treatment outcomes for brachytherapy in disease sites, such as the prostate and cervix. However, newer data suggest that this trend may be reversing. The renewed call for shorter radiation courses based on data showing equivalent outcomes will likely establish hypofractionated radiation as the standard of care across multiple disease sites. With shifting reimbursement, brachytherapy represents the pinnacle in hypofractionated, conformal radiation therapy, and with extensive long-term data in support of the treatment modality brachytherapy is primed for a renaissance.

Simulation-based learning for enhanced gynecologic brachytherapy training among radiation oncology residents.

PURPOSE: Brachytherapy is an irreplaceable component of gynecologic cancer treatment. Resident training has declined, and procedural exposure is variable. We evaluated whether simulation-based gynecologic brachytherapy training among radiation oncology residents could improve knowledge, confidence, and interest. METHODS AND MATERIALS: Before a brachytherapy workshop, radiation oncology residents without prior gynecologic brachytherapy experience completed a survey on brachytherapy knowledge, procedural confidence, plan evaluation, and quality/safety. Residents then participated in a gynecologic brachytherapy workshop. Lectures covered brachytherapy imaging and physics principles/quality assurance, followed by hands-on and individualized feedback regarding applicator selection and placement, target segmentation, and physics quality assurance. Afterward, preworkshop questions were recollected. Descriptive statistics and Fisher's exact tests were used for data analysis. RESULTS: After the workshop, resident responses regarding the learning environment and baseline knowledge questions improved overall. There was a 30% improvement in favorable responses to the learning environment statement "My residency has a formal process/curriculum to teach brachytherapy" and for baseline knowledge the greatest improvement was seen for "I am familiar with the anatomy and placement of the applicators in relation to the anatomy". "Lack of didactic or procedural training exposure" was identified as the main reason for declining brachytherapy use. Initially, 1/8 residents correctly completed the knowledge questions, and after the workshop, 6/7 (p < 0.001) residents correctly completed the questions. CONCLUSIONS: Domain-specific knowledge, procedural confidence, and brachytherapy interest improved after a gynecologic brachytherapy workshop. Integrated didactic and simulation-based brachytherapy training may serve as a valuable learning tool to augment resident knowledge, introduce practical skills, and spark resident interest in brachytherapy.

COVID-19 impact on timing of brachytherapy treatment and strategies for risk mitigation.

PURPOSE: The purpose of this study was to highlight the importance of timely brachytherapy treatment for patients with gynecologic, breast, and prostate malignancies, and provide a framework for brachytherapy clinical practice and management in response to the COVID-19 pandemic. METHODS AND MATERIALS: We review amassing evidence to help guide the management and timing of brachytherapy for gynecologic, breast, and prostate cancers. Where concrete data could not be found, peer-reviewed expert opinion is provided. RESULTS: There may be a significant negative impact on oncologic outcomes for patients with gynecologic malignancies who have a delay in the timely completion of therapy. Delay of prostate or breast cancer treatment may also impact oncologic outcomes. If a treatment delay is expected, endocrine therapy may be an appropriate temporizing measure before delivery of radiation therapy. The use of shorter brachytherapy fractionation schedules will help minimize patient exposure and conserve resources. CONCLUSIONS: Brachytherapy remains a critical treatment for patients and may shorten treatment time and exposure for some. Reduced patient exposure and resource utilization is important during COVID-19. Every effort should be made to ensure timely brachytherapy delivery for patients with gynecologic malignancies, and endocrine therapy may help temporize treatment delays for breast and prostate cancer patients. Physicians should continue to follow developing institutional, state, and federal guidelines/recommendations as challenges in delivering care during COVID-19 will continue to evolve.

Chronic oxidative stress increases the integration frequency of foreign DNA and human papillomavirus 16 in human keratinocytes.

Cervical cancer is the second most common cancer, and the fourth most common cause of cancer death in women worldwide. Nearly all of these cases are caused by high-risk HPVs (HR HPVs), of which HPV16 is the most prevalent type. In most cervical cancer specimens, HR HPVs are found integrated into the human genome, indicating that integration is a key event in cervical tumor development. An understanding of the mechanisms that promote integration may therefore represent a unique opportunity to intercept carcinogenesis. To begin identifying these mechanisms, we tested the hypothesis that chronic oxidative stress (OS) induced by virus- and environmentallymediated factors can induce DNA damage, and thereby increase the frequency with which HPV integrates into the host genome. We found that virus-mediated factors are likely involved, as expression of E6*, a splice isoform of HPV16 E6, increased the levels of reactive oxygen species (ROS), caused oxidative DNA damage, and increased the frequency of plasmid DNA integration as assessed by colony formation assays. To assess the influence of environmentally induced chronic OS, we used L-Buthionine-sulfoximine (BSO) to lower the level of the intracellular antioxidant glutathione. Similar to our observations with E6*, glutathione depletion by BSO also increased ROS levels, caused oxidative DNA damage and increased the integration frequency of plasmid DNA. Finally, under conditions of chronic OS, we were able to induce and characterize a few independent events in which episomal HPV16 integrated into the host genome of cervical keratinocytes. Our results support a chain of events leading from induction of oxidative stress, to DNA damage, to viral integration, and ultimately to carcinogenesis.

Cellular levels of oxidative stress affect the response of cervical cancer cells to chemotherapeutic agents.

Treatment of advanced and relapsed cervical cancer is frequently ineffective, due in large part to chemoresistance. To examine the pathways responsible, we employed the cervical carcinoma-derived SiHa and CaSki cells as cellular models of resistance and sensitivity, respectively, to treatment with chemotherapeutic agents, doxorubicin, and cisplatin. We compared the proteomic profiles of SiHa and CaSki cells and identified pathways with the potential to contribute to the differential response. We then extended these findings by comparing the expression level of genes involved in reactive oxygen species (ROS) metabolism through the use of a RT-PCR array. The analyses demonstrated that the resistant SiHa cells expressed higher levels of antioxidant enzymes. Decreasing or increasing oxidative stress led to protection or sensitization, respectively, in both cell lines, supporting the idea that cellular levels of oxidative stress affect responsiveness to treatment. Interestingly, doxorubicin and cisplatin induced different profiles of ROS, and these differences appear to contribute to the sensitivity to treatment displayed by cervical cancer cells. Overall, our findings demonstrate that cervical cancer cells display variable profiles with respect to their redox-generating and -adaptive systems, and that these different profiles have the potential to contribute to their responses to treatments with chemotherapy.

Human papillomavirus type 16 E6* induces oxidative stress and DNA damage.

UNLABELLED: High-risk types of human papillomavirus (HPV) are the causative agents of virtually all cases of cervical cancer and a significant proportion of other anogenital cancers, as well as both oral and pharyngeal cancers. The high-risk types encode two viral oncogenes, E6 and E7, which work together to initiate cell transformation. Multiple steps involving the activities and interactions of both viral and cellular proteins are involved in the progression from HPV infection to cell transformation to cancer. The E6 oncoprotein is expressed as several isoforms: a full-length variant referred to as E6 and a few shorter isoforms collectively referred to as E6*. In this study, we found that expression of E6* increased the level of reactive oxygen species (ROS) in both HPV-positive and HPV-negative cells. This increased oxidative stress led to higher levels of DNA damage, as assessed by the comet assay, quantification of 8-oxoguanine, and poly(ADP-ribose) polymerase 1. The observed increase in ROS may be due to a decrease in cellular antioxidant activity, as we found that E6* expression also led to decreased expression of superoxide dismutase isoform 2 and glutathione peroxidase. These studies indicate that E6* may play an important role in virus-induced mutagenesis by increasing oxidative stress and DNA damage. IMPORTANCE: Our findings demonstrate for the first time that an HPV gene product, E6*, can increase ROS levels in host cells. This ability may play a significant role both in the viral life cycle and in cancer development, because an increase in oxidative DNA damage may both facilitate HPV genome amplification and increase the probability of HPV16 DNA integration. Integration, in turn, is thought to be an important step in HPV-mediated carcinogenesis.

The small splice variant of HPV16 E6, E6, reduces tumor formation in cervical carcinoma xenografts.

High-risk types of human papillomavirus (HPV) cause nearly all cases of cervical cancer. The E6 oncoprotein is produced as a full-length variant (E6) as well as several shorter isoforms (E6). E6 inhibits certain oncogenic activities of E6, suggesting that it might play an anti-oncogenic role in vivo. To test this, we created E6-expressing SiHa (HPV(+)) and C33A (HPV(-)) cells, then examined the ability of both the parental and E6-expressing cells to form tumors in nude mice. We found that over-expression of E6 indeed decreased the growth of tumors derived from both SiHa and C33A cells, with the reduction greatest in tumors derived from E6-expressing SiHa cells. These findings point to multiple anti-oncogenic characteristics of E6, some of which likely involve down-regulation of the full-length isoform, and others that are independent of HPV. These data represent the first demonstration of biologically-relevant E6 activities distinct from those of the full-length isoform in vivo.

HPV-DNA integration and carcinogenesis: putative roles for inflammation and oxidative stress.

HPV-DNA integration into cellular chromatin is usually a necessary event in the pathogenesis of HPV-related cancer; however, the mechanism of integration has not been clearly defined. Breaks must be created in both the host DNA and in the circular viral episome for integration to occur, and studies have shown that viral integration is indeed increased by the induction of DNA double strand breaks. Inflammation generates reactive oxygen species, which in turn have the potential to create such DNA strand breaks. It is plausible that these breaks enable a greater frequency of HPV-DNA integration, and in this way contribute to carcinogenesis. Consistent with this idea, co-infections with certain sexually transmitted diseases cause cervical inflammation, and have also been identified as cofactors in the progression to cervical cancer. This article examines the idea that inflammation facilitates HPV-DNA integration into cellular chromatin through the generation of reactive oxygen species, thereby contributing to carcinogenesis.