Preclinical Models of Anal Cancer Combined-Modality Therapy.

INTRODUCTION: There have been no significant changes in anal cancer treatment options in 4 decades. In this study, we highlight two preclinical models designed to assess anal cancer treatments. MATERIALS AND METHODS: Transgenic K14E6/E7 mice were treated with 7, 12-dimethylbenz(a)anthracene until anal tumors developed. Mice were treated with localized radiation in addition to chemotherapy (combined-modality therapy [CMT]) and compared to no treatment control (NTC). K14E6/E7 mouse anal spheroids with and without Pik3ca mutations were isolated and treated with vehicle, LY3023414 (LY3) (a drug previously shown to be effective in cancer prevention), CMT, or CMT + LY3. RESULTS: In the in vivo model, there was a significant increase in survival in the CMT group compared to the NTC group (P = 0.0392). In the ex vivo model, there was a significant decrease in the mean diameter of CMT and CMT + LY3-treated spheroids compared to vehicle (P ≤ 0.0001). For LY3 alone compared to vehicle, there was a statistically significant decrease in spheroid size in the K14E6/E7 group without mutation (P = 0.0004). CONCLUSIONS: We have provided proof of concept for two preclinical anal cancer treatment models that allow for the future testing of novel therapies for anal cancer.

Emerging Prognostic and Predictive Significance of Stress Keratin 17 in HPV-Associated and Non HPV-Associated Human Cancers: A Scoping Review.

A growing body of literature suggests that the expression of cytokeratin 17 (K17) correlates with inferior clinical outcomes across various cancer types. In this scoping review, we aimed to review and map the available clinical evidence of the prognostic and predictive value of K17 in human cancers. PubMed, Web of Science, Embase (via Scopus), Cochrane Central Register of Controlled Trials, and Google Scholar were searched for studies of K17 expression in human cancers. Eligible studies were peer-reviewed, published in English, presented original data, and directly evaluated the association between K17 and clinical outcomes in human cancers. Of the 1705 studies identified in our search, 58 studies met criteria for inclusion. Studies assessed the prognostic significance (n = 54), predictive significance (n = 2), or both the prognostic and predictive significance (n = 2). Altogether, 11 studies (19.0%) investigated the clinical relevance of K17 in cancers with a known etiologic association to HPV; of those, 8 (13.8%) were focused on head and neck squamous cell carcinoma (HNSCC), and 3 (5.1%) were focused on cervical squamous cell carcinoma (SCC). To date, HNSCC, as well as triple-negative breast cancer (TNBC) and pancreatic cancer, were the most frequently studied cancer types. K17 had prognostic significance in 16/17 investigated cancer types and 43/56 studies. Our analysis suggests that K17 is a negative prognostic factor in the majority of studied cancer types, including HPV-associated types such as HNSCC and cervical cancer (13/17), and a positive prognostic factor in 2/17 studied cancer types (urothelial carcinoma of the upper urinary tract and breast cancer). In three out of four predictive studies, K17 was a negative predictive factor for chemotherapy and immune checkpoint blockade therapy response.

A phase I study of intra-anal artesunate (suppositories) to treat anal high-grade squamous intraepithelial lesions.

BACKGROUND: Ablation or surgical excision is the typical treatment of anal high-grade squamous intraepithelial lesions (HSIL). Recurrences are common due to the persistence of underlying human papillomavirus (HPV) infection. Additional well-tolerated and effective non-surgical options for HPV-associated anal disease are needed. METHODS: This 3+3 dose escalation Phase I clinical trial evaluated the safety and tolerability of artesunate suppositories in the treatment of patients with biopsy-proven HSIL. RESULTS: The maximal tolerated dose was 400 mg, administered in 3 cycles. All adverse events associated with the use 200- and 400-mg artesunate suppositories were Grade 1. At the 600-mg dose, patients experienced clinically significant nausea. CONCLUSION: Artesunate suppositories are a safe treatment option for anal HSIL.

Topical Protease Inhibitor Decreases Anal Carcinogenesis in a Transgenic Mouse Model of HPV Anal Disease.

Anal cancer is a major health problem. This study seeks to determine if the topical protease inhibitor Saquinavir (SQV), is effective at the prevention of anal cancer in transgenic mice with established anal dysplasia. K14E6/E7 mice were entered into the study when the majority spontaneously developed high-grade anal dysplasia. To ensure carcinoma development, a subset of the mice was treated with a topical carcinogen: 7,12-Dimethylbenz[a]anthracene (DMBA). Treatment groups included: no treatment, DMBA only, and topical SQV with/without DMBA. After 20 weeks of treatment, anal tissue was harvested and evaluated histologically. SQV was quantified in the blood and anal tissue, and tissue samples underwent analysis for E6, E7, p53, and pRb. There was minimal systemic absorption of SQV in the sera despite high tissue concentrations. There were no differences in tumor-free survival between SQV-treated and respective control groups but there was a lower grade of histological disease in the mice treated with SQV compared to those untreated. Changes in E6 and E7 levels with SQV treatment suggest that SQV may function independently of E6 and E7. Topical SQV decreased histological disease progression in HPV transgenic mice with or without DMBA treatment without local side effects or significant systemic absorption.

The Use of the Protease Inhibitor, Saquinavir, to Treat Anal Cancer Spheroids Derived From Human Papillomavirus Transgenic Mice.

BACKGROUND: Anal cancer is associated with high-risk human papillomavirus infection and oncoprotein expression. We have identified several protease inhibitors, used to treat HIV, that decrease oncogene expression. OBJECTIVE: The aim of this project is to determine whether saquinavir, a protease inhibitor, results in a treatment response in anal cancer spheroids. DESIGN: K14E6/E7 transgenic mice (n = 5), which express human papillomavirus 16 oncoproteins E6 and E7 in their epithelium, were treated topically at the anus with a carcinogen, 7,12-dimethylbenz[a]anthracene, to promote anal tumor growth. Tumors were excised and digested, and cells were plated. The tumor cells form 3D multicellular aggregates known as spheroids. SETTINGS: This study was performed in an American Association for Accreditation of Laboratory Animal Care-approved facility. INTERVENTIONS: Spheroids were placed in treatment groups: no treatment, vehicle (dimethyl sulfoxide), and 15 µM saquinavir. Spheroids were imaged immediately pretreatment and 24 hours posttreatment. MAIN OUTCOME MEASURES: Spheroid diameters were measured using ImageJ and mean percent reduction was calculated for each spheroid to determine treatment effect on spheroid growth. Analysis of variance using pairwise comparisons was performed with Fisher protected least significant difference tests. RESULTS: The no-treatment (n = 119 spheroids) and vehicle (n = 126 spheroids) groups demonstrated an increase in spheroid diameter during the treatment period. In contrast, spheroids treated with saquinavir (n = 151 spheroids) demonstrated a statistically significant percent reduction compared to the no-treatment ( p < 0.0001) and vehicle ( p = 0.002) groups. LIMITATIONS: A limitation of these data is that some human error is likely present given that images were analyzed by 3 different scientists. CONCLUSIONS: Saquinavir leads to a statistically significant percent reduction in mice anal tumor spheroid growth ex vivo compared to control groups. Protease inhibitor therapy may be an effective treatment or adjuvant therapy to the Nigro protocol to promote anal cancer tumor regression. See Video Abstract at http://links.lww.com/DCR/C82 . EL USO DEL INHIBIDOR DE LA PROTEASA, SAQUINAVIR, PARA TRATAR LOS ESFEROIDES DEL CNCER ANAL DERIVADOS DE RATONES TRANSGNICOS PARA EL VPH: ANTECEDENTES:El cáncer anal está asociado con la infección por el virus del papiloma humano de alto riesgo y la expresión de oncoproteínas. Hemos identificado varios inhibidores de la proteasa, utilizados para tratar el VIH, que disminuyen la expresión del oncogén.OBJETIVO:El objetivo de este proyecto es determinar si los esferoides de cáncer anal responden al tratamiento con inhibidor de la proteasa, Saquinavir.DISEÑO:Ratones transgénicos K14E6/E7 (n = 5), que expresan las oncoproteínas E6 y E7 del VPH16 en su epitelio, fueron tratados tópicamente en el ano con carcinógeno, 7,12 dimetilbenz[a]antraceno, para promover el crecimiento del tumor anal. Los tumores se extirparon y digirieron, y las células se sembraron en placas. Las células tumorales forman agregados multicelulares tridimensionales, conocidos como esferoides.ESCENARIO:Este estudio se realizó en un centro aprobado por la Asociación Estadounidense para la Acreditación de Cuidado de Animales de Laboratorio.INTERVENCIONES:Se colocaron esferoides en grupos de tratamiento: sin tratamiento, vehículo (sulfóxido de dimetilo) y saquinavir 15 µM. Se tomaron imágenes de los esferoides inmediatamente antes del tratamiento y 24 horas después del tratamiento.PRINCIPALES MEDIDAS DE RESULTADO:Los diámetros de los esferoides se midieron con ImageJ y se calculó el porcentaje medio de reducción de cada esferoide para determinar el efecto del tratamiento sobre el crecimiento de los esferoides. El análisis de varianza mediante comparaciones por pares se realizó con las pruebas de diferencia mínima significativa protegida de Fisher.RESULTADOS:Los grupos sin tratamiento (n =119 esferoides) y vehículo (n=126 esferoides) demostraron un aumento en el diámetro del esferoide durante el período de tratamiento. Por el contrario, los esferoides tratados con saquinavir (n =151 esferoides) demostraron una reducción porcentual estadísticamente significativa en comparación con los grupos sin tratamiento ( p < 0,0001) y con vehículo (p = 0,002).LIMITACIONES:una limitación de estos datos es que es probable que haya algún error humano dado que las imágenes fueron analizadas por tres científicos diferentes.CONCLUSIONES:Saquinavir conduce a una reducción porcentual estadísticamente significativa en el crecimiento de esferoides de tumores anales en ratones ex-vivo en comparación con los grupos de control. La terapia con inhibidores de la proteasa puede ser un tratamiento eficaz o una terapia adyuvante del protocolo Nigro para promover la regresión del tumor del cáncer anal. Consulte Video Resumen en http://links.lww.com/DCR/C82 . (Traducción-Dr. Felipe Bellolio ).

Anal Cancer Prevention Through the Topical Use of Single or Dual PI3K/mTOR Inhibitors.

INTRODUCTION: Anal dysplasia and anal cancer are major health problems. This study seeks to determine if inhibition of mTOR and/or PI3K pathways is effective at anal cancer prevention in mice with/without established precancerous lesions of the anus (anal dysplasia). METHODS: K14E6/E7 mice were entered into the study at 5 wk, 15 wk, or 25 wk of age. Mice were treated with a topical carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA), which ensures carcinoma development within 20 wk. Treatment groups included: no treatment, DMBA only, topical Pictilisib (PI3K inhibitor) with/without DMBA, topical Sapanisertib (mTOR inhibitor) with/without DMBA, and topical Samotolisib (dual PI3K/mTOR inhibitor) with/without DMBA. Mice underwent weekly observations for anal tumor development (tumor-free survival). After 20 wk of treatment, anal tissue was harvested and evaluated histologically for squamous cell carcinoma (SqCC). RESULTS: All topical treatments in conjunction with DMBA increased tumor-free survival in mice that started treatment at 15 wk of age when compared to DMBA-only treatment, except for Pictilisib + DMBA in males. Topical Sapanisertib increased tumor-free survival in mice regardless of starting treatment age. When examining tissue for microscopic evidence of SqCC, only topical Samotolisib in males decreased SqCC in the 15 wk starting mice. CONCLUSIONS: Sapanisertib, the mTOR inhibitor, had the greatest effect, in terms of increasing tumor-free survival, regardless of starting time point or sex. Unlike the other treatments, Samotolisib, the dual PI3K/mTOR inhibitor, decreased microscopic evidence of SqCC when starting treatment at 15 wk of age but only in male mice.

The use of a topical protease inhibitor, Saquinavir, to alleviate mouse papillomavirus-mediated anal disease.

Select protease inhibitors (PI) have been found to be effective in decreasing human papillomavirus oncoprotein expression. This study evaluated whether the topical PI, Saquinavir (SQV), promotes viral clearance in an infectious mouse model with Mus musculus papillomavirus 1 (MmuPV1). NOD scid gamma (NSG) mice were anally infected with ∼4 × 108 viral genome equivalents of MmuPV1 and 120 days post-infection (when majority have high-grade anal dysplasia), began topical treatments: control (mock), 7,12-dimethylbenz(a)anthracene (DMBA) only, once weekly to promote carcinogenesis, 1% SQV only, daily (Monday - Friday), and SQV + DMBA. Viral MmuPV1 load was analyzed from anal lavages pre and post-treatment. Anal tissue was harvested, processed, and evaluated for drug absorption, grade of anal disease, and anal viral RNA. Results suggest that topical SQV promotes decreased viral shedding in female mice treated with SQV.

Efficacy of Topically Administered Dihydroartemisinin in Treating Papillomavirus-Induced Anogenital Dysplasia in Preclinical Mouse Models.

The artemisinin family of compounds is cytopathic in certain cancer cell lines that are positive for human papillomaviruses (HPV) and can potentially drive the regression of dysplastic lesions. We evaluated the efficacy of topical dihydroartemisinin (DHA) on cervical dysplasia and anal dysplasia in two papillomavirus mouse models: K14E6/E7 transgenic mice, which express HPV16 oncogenes; and immunodeficient NOD/SCID gamma (NSG) mice infected with Mus musculus papillomavirus (MmuPV1). Mice started treatment with DHA at 25 weeks of age (K14E6/E7) or 20 weeks post infection (MmuPV1-infected), when the majority of mice are known to have papillomavirus-induced low- to high-grade dysplasia. Mice were treated with or without topical DHA at the cervix or anus and with or without topical treatment with the chemical carcinogen 7,12 dimethylbenz(a)anthracene (DMBA) at the anus of in transgenic mice to induce neoplastic progression. Mice were monitored for overt tumor growth, and tissue was harvested after 20 weeks of treatment and scored for severity of histological disease. For MmuPV1-infected mice, anogenital lavages were taken to monitor for viral clearance. Tissues were also evaluated for viral gene expression at the RNA and/or protein levels. Treatment with topical DHA did not reduce dysplasia in the anogenital tract in either papillomavirus-induced mouse model and did not prevent progression to anal cancer in the DMBA-treated K14E6/E7 mice.

PI3K/mTOR inhibition prevents anal cancer in mice with established low-grade anal dysplasia.

Low-grade anal dysplasia is a disease that can progress to high-grade anal dysplasia and eventually anal cancer if left untreated. Research has shown that low-grade anal dysplasia is marked by significant autophagic dysfunction. We hypothesized that systemic induction of autophagy, via phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibition, would be effective in preventing anal cancer development in human papillomavirus (HPV) mice (K14E6/E7) with established low-grade anal dysplasia. Mice began treatment at 15 weeks of age, when 75% of mice spontaneously develop low-grade anal dysplasia, and were divided into the following groups: no treatment, systemic LY3023414 (4.5 mg/kg, dual PI3K/mTOR inhibitor) alone, topical 7,12 dimethylbenz[a]anthracene (DMBA) alone, or systemic LY3023414 and topical DMBA. Groups were compared for final histology, PI3K activity, mTOR activity, autophagic induction (light chain 3B (LC3ß)), autophagic function (p62 protein), and tumor-free survival. Untreated mice or mice treated with LY3023414 alone did not progress to cancer. There was a statistically significant decrease in the number of mice that developed histologic evidence of cancer when comparing mice that received systemic LY3203414 with topical DMBA versus those that received topical DMBA alone (p = 0.0003). PI3K and mTOR activity decreased in groups treated with systemic LY3023414 and topical DMBA as compared with those treated with topical DMBA alone (p = 0.0005 and p = 0.0271, respectively). LC3ß and p62 expression was not statistically altered with systemic LY3023414 treatment. Mice developed less overt tumors and had increased tumor-free survival when treated with systemic LY3023414 in the presence of topical DMBA compared to topical DMBA alone (p = 0.0016 and p < 0.001, respectively). Systemic LY3023414 treatment is effective in anal cancer prevention in the setting of established low-grade anal dysplasia in an HPV-associated mouse model of anal cancer.

Systemic Delivery of a Dual PI3K/mTOR Inhibitor More Effective than Topical Delivery in Preventing Anal Carcinogenesis in an HPV Transgenic Mouse Model.

Introduction: Anal dysplasia is a growing health concern that over time can result in squamous cell carcinoma (SqCC) of the anus. In this study, we compare a topical versus systemic (oral) administration of LY3023414, a dual PI3K/mTOR inhibitor, to prevent anal carcinogenesis in a Human Papillomavirus (HPV) mouse model of anal cancer. Materials and Methods: K14E6/E7 transgenic mice were used to model HPV-induced anal carcinogenesis. Mice with varying starting anal histologies (normal histology, low-grade, and high-grade anal dysplasia) were treated topically at the anus or systemically via oral gavage with LY3023414 with or without topical carcinogen for 20 weeks. Mice were monitored for overt anal tumor development and anal tissue was assessed for histology and markers of PI3K and mTOR activity (pAKT and pS6, respectively). Results: LY3023414 treatment, regardless of the mode of delivery, significantly decreased overt tumor development in mice starting with normal histology and low-grade anal dysplasia. Systemic LY3023414 treatment was more effective in delaying tumor onset than topical treatment. Mice treated with systemic LY3023414 had significantly reduced rates of anal SqCC when starting with normal and low-grade anal dysplasia compared to topical treatment. Topical treatment was only effective in reducing SqCC in the setting of low-grade dysplasia. LY3023414 inhibition of pAKT and pS6 expression varied with starting histology. Neither treatment mode was effective in the setting of high-grade anal dysplasia. Conclusion: Systemic LY3023414 treatment was more effective than topical application in delaying the progression of normal anal histology and low-grade dysplasia to anal cancer in HPV-associated mice.

HIV-1 Protease Inhibitors Slow HPV16-Driven Cell Proliferation through Targeted Depletion of Viral E6 and E7 Oncoproteins.

High-risk human papillomavirus strain 16 (HPV16) causes oral and anogenital cancers through the activities of two viral oncoproteins, E6 and E7, that dysregulate the host p53 and pRb tumor suppressor pathways, respectively. The maintenance of HPV16-positive cancers requires constitutive expression of E6 and E7. Therefore, inactivating these proteins could provide the basis for an anticancer therapy. Herein we demonstrate that a subset of aspartyl protease inhibitor drugs currently used to treat HIV/AIDS cause marked reductions in HPV16 E6 and E7 protein levels using two independent cell culture models: HPV16-transformed CaSki cervical cancer cells and NIKS16 organotypic raft cultures (a 3-D HPV16-positive model of epithelial pre-cancer). Treatment of CaSki cells with some (lopinavir, ritonavir, nelfinavir, and saquinavir) but not other (indinavir and atazanavir) protease inhibitors reduced E6 and E7 protein levels, correlating with increased p53 protein levels and decreased cell viability. Long-term (>7 day) treatment of HPV16-positive NIKS16 raft cultures with saquinavir caused epithelial atrophy with no discernible effects on HPV-negative rafts, demonstrating selectivity. Saquinavir also reduced HPV16's effects on markers of the cellular autophagy pathway in NIKS16 rafts, a hallmark of HPV-driven pre-cancers. Taken together, these data suggest HIV-1 protease inhibitors be studied further in the context of treating or preventing HPV16-positive cancers.

Preoperative Tamsulosin to Prevent Postoperative Urinary Retention: A Randomized Controlled Trial.

BACKGROUND: The purpose of this study was to evaluate the efficacy of tamsulosin, administered preoperatively, for the prevention of postoperative urinary retention (POUR). POUR is a common complication of abdominal surgery, leading to the use of urinary catheters, which are a risk factor for urinary tract infection. Tamsulosin is a uroselective alpha-1a blocker used for the treatment of lower urinary tract symptoms. MATERIALS AND METHODS: A randomized, double-blind, placebo-controlled trial was undertaken from August 2015 to May 2018. Adults undergoing elective inpatient abdominal surgery were randomized to receive either tamsulosin 0.4 mg or placebo daily for 7 d before surgery and continuing for up to 7 d postoperatively. The primary outcome was need for at least a single intermittent catheterization postoperatively. Secondary outcomes included first postvoid residual volume, number of catheterizations, need for replacement of an indwelling catheter, hospital length of stay, and urinary tract infection within 30 d of surgery. RESULTS: A total of 158 participants were enrolled, with a final analytic cohort of 141 participants. The two groups had similar baseline characteristics, operative characteristics, and timing of catheter removal. There was no difference in the incidence of POUR between the two groups (26% in tamsulosin versus 31% in placebo, P = 0.49). There was also no difference in any of the secondary outcomes between the two groups. Epidural usage, open surgery, and age <50 were identified as risk factors for POUR. CONCLUSIONS: Perioperative prophylaxis with tamsulosin is not effective in reducing the incidence of POUR in patients undergoing elective abdominal surgery.

The Optimal Ileal Pouch: Physiologic Parameters Significantly Correlate with Overall Pouch Function.

BACKGROUND: Ileal pouch function is affected by several patient factors and pouch physiology. The significance of pouch physiology on optimal pouch function has not been well characterized. The purpose of this study was to examine specific post-ileal pouch anal anastomosis (IPAA) physiologic parameters to determine impact on pouch function and quality of life. METHODS: Patients undergoing proctocolectomy with IPAA for ulcerative colitis were examined. Post-IPAA compliance, pouch anal pressure gradient (PAPG), and function were assessed 6-8 months postoperatively. Compliance was calculated as change in volume divided by change in pressure. PAPG was calculated as the difference between anal pressure and intra-pouch pressure at a fixed volume. Pouch function evaluation included stool frequency and episodes of incontinence. Quality of life was evaluated using the Rockwood Fecal Incontinence Quality of Life Scale. RESULTS: A total of 125 patients were investigated. Post-IPAA resting anal pressure averaged 58.1 ± 15 mmHg. Mean volume and intra-pouch pressure at evacuation were 245 mL and 33.9 mmHg, respectively. Compliance averaged 11.2 mmHg/mL with a mean PAPG of - 29.3 mmHg. Compliance and PAPG correlated with 24-h (p = 0.003, p = 0.004) and nighttime stool frequency (p = 0.04, p = 0.03). Daytime continence was impacted by compliance (p = 0.04), PAPG (p = 0.02), and resting anal pressure (p = 0.02). CONCLUSION: This unique evaluation reveals a significant correlation between IPAA physiologic properties and function. Optimal function and quality of life depend in part on maintaining optimal pouch compliance and pressure differentials between the pouch and anal canal, defined by the pouch anal pressure gradient.

Anal Intraepithelial Neoplasia Screening With Anal Pap Tests: Follow-up and Corresponding Histology.

BACKGROUND: Anal cytology is used as a screening tool in the detection of precancerous anal squamous lesions. Follow-up clinical examination after abnormal anal cytology is recommended. The objective of this study was to determine how often abnormal cytology was followed by a clinical examination at our institution and how often cytology predicted histologic outcome. MATERIALS AND METHODS: A retrospective chart review was performed (2008-2018) on patients with anal cytology, demonstrating either low-grade or high-grade squamous intraepithelial lesion. Clinical examination within 1 y (digital rectal examination, anoscopy, or high-resolution anoscopy) was recorded. The probability of anal intraepithelial neoplasm on biopsy after dysplasia on cytology was calculated, and McNemar's test was used to determine if there was correspondence between cytology and histology. RESULTS: A total of 327 anal cytology results demonstrated dysplasia (75% low grade and 25% high grade) in 182 patients. Seventy-five percent of dysplastic anal cytology were followed by clinical examination within 1 y, and 50% were biopsied. The probability of dysplasia on histology after dysplasia on cytology was 72% (95% confidence interval: 64%-78.5%). Twenty-eight percent of low-grade cytology results were upgraded to advanced disease (high-grade or invasive cancer) on histology. A low-grade cytology result was unable to preclude high-grade histology in our population. CONCLUSIONS: There is room for improvement at our institution to consistently follow-up with clinical examination after abnormal anal cytology. Our data suggest this is especially important considering anal cytology is an imperfect predictor of histologic anal intraepithelial neoplasia and invasive disease. Clinical examination is a critical component of anal dysplasia screening and follow-up.

Patient-Derived Cancer Organoid Cultures to Predict Sensitivity to Chemotherapy and Radiation.

PURPOSE: Cancer treatment is limited by inaccurate predictors of patient-specific therapeutic response. Therefore, some patients are exposed to unnecessary side effects and delays in starting effective therapy. A clinical tool that predicts treatment sensitivity for individual patients is needed. EXPERIMENTAL DESIGN: Patient-derived cancer organoids were derived across multiple histologies. The histologic characteristics, mutation profile, clonal structure, and response to chemotherapy and radiation were assessed using bright-field and optical metabolic imaging on spheroid and single-cell levels, respectively. RESULTS: We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. These cultures were generated from numerous cancers, various biopsy sample types, and in different clinical settings. Next-generation sequencing reveals the presence of subclonal populations within the organoid cultures. These cultures allow for the detection of clonal heterogeneity with a greater sensitivity than bulk tumor sequencing. Optical metabolic imaging of these organoids provides cell-level quantification of treatment response and tumor heterogeneity allowing for resolution of therapeutic differences between patient samples. Using this technology, we prospectively predict treatment response for a patient with metastatic colorectal cancer. CONCLUSIONS: These studies add to the literature demonstrating feasibility to grow clinical patient-derived organotypic cultures for treatment effectiveness testing. Together, these culture methods and response assessment techniques hold great promise to predict treatment sensitivity for patients with cancer undergoing chemotherapy and/or radiation.

Topical application of a dual PI3K/mTOR inhibitor prevents anal carcinogenesis in a human papillomavirus mouse model of anal cancer.

Human papillomavirus (HPV) infection is the major risk factor for anal dysplasia that may progress to squamous cell carcinoma of the anus. We have previously shown that systemic administration of a PI3K/mTOR inhibitor (BEZ235), an autophagic inducer, results in decreased squamous cell carcinoma of the anus in our HPV mouse model. In this study, we investigate the effect of the local, topical application of a BEZ235 on tumor-free survival, histopathology, PI3K/mTOR, and autophagy. The rationale for investigating a topical formulation is the localized nature of anal dysplasia/cancer and the goal for creating a clinically translatable formulation to decrease anal carcinogenesis. In this study, HPV transgenic mice were given no treatment, topical BEZ235, topical 7,12 dimethylbenz[a]anthracene (DMBA) (carcinogen), or both topical DMBA + BEZ235. Mice were assessed for tumor development and treatment-related toxicities. Tissue was evaluated for histology, PI3K/mTOR inhibition (pS6 and pAkt), and autophagy (LC3ß and p62). DMBA-alone mice had an average of 16.9 weeks tumor-free survival, whereas mice receiving both DMBA+topical BEZ235 had 19.3 weeks (P < 0.000001). Histopathology revealed a significant decrease in dysplasia/carcinoma with the addition of topical BEZ235 to DMBA (P < 0.000001). Comparing DMBA versus DMBA + BEZ235, topical BEZ235 resulted in a significant decrease in both pS6 and pAkt (P < 0.001). Compared with no-treatment mice, both BEZ235-treated and DMBA + BEZ235-treated mice had significantly higher LC3ß expression, signifying autophagic induction (P < 0.01), whereas DMBA-treated, BEZ235-treated, and DMBA+BEZ235-treated mice had a significantly lower p62 expression, signifying active autophagy (P < 0.0005). In conclusion, consistent with systemic delivery, topical application of BEZ235 shows decreased anal carcinogenesis through the activation of autophagy.

Genetic inhibition of autophagy in a transgenic mouse model of anal cancer.

BACKGROUND: The dynamic role of autophagy in cancer development is a topic of considerable research and debate. Previously published studies have shown that anal cancer development can be promoted or prevented with the pharmacologic inhibition or induction, respectively, of autophagy in a human papillomavirus (HPV) mouse model. However, these results are confounded by the fact that the drugs utilized are known to affect other pathways besides autophagy. It has also been shown that autophagic inhibition occurs in the setting of HPV16 oncoprotein expression (E6 and E7) and correlates with increased susceptibility to anal carcinogenesis. MATERIALS AND METHODS: In this study, we employed a conditional, genetic, autophagic (Atg7) knockout mouse model to determine conclusively that autophagy has a role in anal cancer development, in the absence or presence of E6 and E7. RESULTS: In mice lacking both HPV16 oncogenes, knockout of autophagy followed by exposure to a carcinogen resulted in a tumor incidence of 40%, compared to 0% in mice treated with a carcinogen alone with an intact autophagic pathway (P = 0.007). In mice expressing either one or both HPV16 oncoproteins, the addition of genetic knockout of autophagy to carcinogen treatment did not lead to a significant difference in tumor incidence compared to carcinogen treatment alone, consistent with the ability of HPV oncogenes to inhibit autophagy in themselves. CONCLUSIONS: These results provide the first conclusive evidence for the distinct role of autophagy in anal carcinogenesis, and suggest that autophagy is a plausible target for therapies aimed at reducing anal dysplasia and anal cancer development.

Defecography: An Overview of Technique, Interpretation, and Impact on Patient Care.

Pelvic floor and defecatory dysfunction are common in the female patient population. When combined with physical examination, barium defecography allows for accurate and expanded assessment of the underlying pathology and helps to guide future intervention. Understanding the imaging findings of barium defecography in the spectrum of pathology of the anorectum and pelvic floor allows one to appropriately triage and treat patients presenting with defecatory dysfunction.

Clinically Competent and Fiscally at Risk: Impact of Debt and Financial Parameters on the Surgical Resident.

BACKGROUND: While the costs of medical training continue to increase, surgeon income and personal financial decisions may be challenged to manage this expanding debt burden. We sought to characterize the financial liability, assets, income, and debt of surgical residents, and evaluate the necessity for additional financial training. STUDY DESIGN: All surgical trainees at a single academic center completed a detailed survey. Questions focused on issues related to debt, equity, cash flow, financial education, and fiscal parameters. Responses were used to calculate debt-to-asset and debt-to-income ratios. Predictors of moderate risk debt-to-asset ratio (0.5 to 0.9), high risk debt-to-asset ratio (≥0.9), and high risk debt-to-income ratio (>0.4) were evaluated. All analyses were performed in SPSS v.21. RESULTS: One hundred five trainees completed the survey (80% response rate), with 38% of respondents reporting greater than $200,000 in educational debt. Overall, 82% of respondents had a moderate or high risk debt-to-asset ratio. Residency program, year, sex, and perception of financial knowledge did not correlate with high risk debt-to-asset ratio. Residents with high debt-to-asset ratios were more likely to have a high level of concern about debt (52% vs 0%, p < 0.001) when compared with residents who had low debt-to-asset ratios. The majority (79%) of respondents felt strongly that inclusion of additional financial training in residency education is a critical need. CONCLUSIONS: In a climate of increasingly delayed financial gratification, surgical trainees are on critically unstable financial footing. There is a major gap in current surgical education that requires reassessment for the long-term financial health of residents.