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.

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.

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.

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.

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.

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.

Does Anastomotic Leak Contribute to High Failure-to-rescue Rates?

OBJECTIVE: Our aim was to evaluate failure-to-rescue (FTR) after anastomotic leak (AL) in colectomy patients. BACKGROUND: In the era of pay for performance, it is imperative that we understand the quality measures under which we are scrutinized. FTR has been proposed as a marker of surgical quality. We investigated the role of complications in FTR rates in colectomy patients. METHODS: Patients who underwent nonemergent colectomy from 2012 to 2013 were identified from the The American College of Surgeons National Quality Improvement Program (ACS NSQIP database). Mortality after AL was assessed and stratified in relation to mortality after other postoperative complications. χ and logistic regression analysis were used to assess the effect of AL on mortality. RESULTS: We identified 30,101 patients who met inclusion criteria, 1127 suffered an AL (3.7%). FTR was increased in patients with AL compared with those without AL (6% vs 1%, P < 0.001). The mortality rate after leak was similar to mortality after other major complications. Independent risk factors for death after AL included older age (odds ratio [OR] 3.140; 95% confidence interval [CI], 1.744-5.651), cancer diagnosis (OR 2.032; 95% CI, 1.177-3.507), and open approach (OR 2.124; 95% CI, 1.194-3.776) while preoperative bowel preparation was protective (OR 0.563; 95% CI, 0.328-0.969). CONCLUSIONS: AL is a common complication after colectomy with a relatively high FTR rate. As hospitals are penalized for not reaching specific rates of FTR, we must better understand these complex relationships to improve quality and safety of patient care.

Short-term outcomes of minimally invasive versus open colectomy for colon cancer.

BACKGROUND: Laparoscopic and open approaches to colon resection have equivalent long-term outcomes and oncologic integrity for the treatment of colon cancer. Differences in short-term outcomes should therefore help to guide surgeons in their choice of operation. We hypothesized that minimally invasive colectomy is associated with superior short-term outcomes compared to traditional open colectomy in the setting of colon cancer. MATERIALS AND METHODS: Patients undergoing nonemergent colectomy for colon cancer in 2012 and 2013 were selected from the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) targeted colectomy participant use file. Patients were divided into two cohorts based on operative approach-open versus minimally invasive surgery (MIS). Univariate, multivariate, and propensity-adjusted multivariate analyses were performed to compare postoperative outcomes between the two groups. RESULTS: A total of 11,031 patients were identified for inclusion in the study, with an overall MIS rate of 65.3% (n = 7200). On both univariate and multivariate analysis, MIS approach was associated with fewer postoperative complications and lower mortality. In the risk-adjusted multivariate analysis, MIS approach was associated with an odds ratio of 0.598 for any postoperative morbidity compared to open (P < 0.001). CONCLUSIONS: This retrospective study of patients undergoing colectomy for colon cancer demonstrates significantly improved outcomes associated with a MIS approach, even when controlling for baseline differences in illness severity. When feasible, minimally invasive colectomy should be considered gold standard for the surgical treatment of colon cancer.

CaMKIα regulates AMP kinase-dependent, TORC-1-independent autophagy during lipopolysaccharide-induced acute lung neutrophilic inflammation.

Autophagy is an evolutionarily conserved cytoplasmic process regulated by the energy rheostats mammalian target of rapamycin and AMP kinase (AMPK) that recycles damaged or unused proteins and organelles. It has been described as an important effector arm of immune cells. We have shown that the cytoplasmically oriented calcium/calmodulin-dependent protein kinase (CaMK)Iα regulates the inflammatory phenotype of the macrophage (M). In this study, we hypothesize that CaMKIα mediates M autophagy. LPS induced autophagy in RAW 264.7 cells and murine peritoneal M that was attenuated with biochemical CaMK inhibition or CaMKIα small interfering RNA (siRNA). Inhibition of CaMKIα reduced LPS-induced p-Thr(172)AMPK and target of rapamycin complex-1 activity, and expression of a constitutively active CaMKIα but not a kinase-deficient mutant induced p-Thr(172)AMPK and autophagy that was attenuated by the AMPK inhibitor compound C. Coimmunoprecipitation and in vitro kinase assays demonstrated that CaMKIα activates AMPK, thereby inducing ATG7, which also localizes to this CaMKIα/AMPK complex. During LPS-induced lung inflammation, C57BL/6 mice receiving CaMKIα(siRNA) displayed reduced lung and bronchoalveolar immune cell autophagy that correlated with reduced neutrophil recruitment, myeloperoxidase activity, and air space cytokine concentration. Independently inhibiting autophagy, using siRNA targeting the PI3K VPS34, yielded similar reductions in lung autophagy and neutrophil recruitment. Thus, a novel CaMKIα/AMPK pathway is rapidly activated in M exposed to LPS and regulates an early autophagic response, independent of target of rapamycin complex-1 inhibition. These mechanisms appear to be operant in vivo in orchestrating LPS-induced lung neutrophil recruitment and inflammation.

Experimental sepsis-induced mitochondrial biogenesis is dependent on autophagy, TLR4, and TLR9 signaling in liver.

Organ injury in sepsis is initially characterized by dysfunction without cell death and structural damage, and thus with the ability to recover organ function. Adaptive metabolic responses to sepsis can prevent bioenergetic failure and death. These studies were aimed at investigating the influence of sepsis on mitochondrial homeostasis, focusing on removal of dysfunctional mitochondria and restitution of a healthy mitochondrial population. These data demonstrate decreased hepatic oxidative phosphorylation by 31 ± 11% following murine cecal ligation and puncture (CLP) at 8 h and 34 ± 9% following LPS treatment in vitro at 12 h (P<0.05). In addition, there was a loss of mitochondrial membrane potential. Mitochondrial density and number initially decreased (relative area per micrograph of 64±10% at baseline vs. 39±13% at 8 h following LPS; P<0.05) and was associated with an increase in autophagy and mitophagy. CLP-induced markers of mitochondrial biogenesis and mitochondrial number and density recovered over time. Furthermore, these data suggest that mitochondrial biogenesis was dependent on an autophagy and mitochondrial DNA/Toll-like receptor 9 (TLR9) signaling pathway. These results suggest that hepatocyte survival and maintenance of function in sepsis is dependent on a mitochondrial homeostasis pathway marked by mitophagy and biogenesis.

Polymicrobial sepsis is associated with decreased hepatic oxidative phosphorylation and an altered metabolic profile.

BACKGROUND: Organ failure in sepsis accounts for significant mortality worldwide. Mitochondrial and metabolic responses are central to the overall response of the cell, and thus of the organ and organism. Adaptive responses in metabolism are critical to the recovery at the cellular level. The purpose of these investigations was to test the hypothesis that sepsis is associated with decreased aerobic respiration and significant metabolic changes in the liver. METHODS: C57BL/6 mice underwent cecal ligation and puncture (CLP) with a 21 gauge needle or an operation without CLP. Mice were euthanized from 0-24 h after the procedure and liver tissue was harvested. Tissue oxygen consumption and mitochondrial complex activity were measured. Global biochemical profiles of 311 metabolites were performed at the 8-h time point (n = 8/group) and analyzed by gas chromatography-mass spectrometry and liquid chromatography tandem mass spectrometry platforms by Metabolon (Durham, North Carolina). The influence of lipopolysaccharide (LPS) on aerobic and anaerobic respiration in primary mouse hepatocytes was also investigated. RESULTS: CLP in vivo or LPS in vitro resulted in a significant decrease in hepatic oxygen consumption. There was a significant decrease in oxidative phosphorylation measured at 12 h. LPS also resulted in a significant increase in anaerobic respiration in hepatocytes. Interestingly, the metabolomic analysis resulted in a metabolic shift in the liver from carbohydrate-based energy to utilization of fatty acids and amino acids. This included an increase in every tricarboxylic acid cycle intermediate and derivative, suggesting an increased flux into the cycle from fatty acid beta-oxidation and anaplerotic contributions from amino acids. CONCLUSIONS: Sepsis results in a metabolic response and profile consistent with increased anaerobic respiration, which occurs prior to significant changes in hemodynamics. The metabolic responses of cells and organs may be important adaptive responses to prevent organ failure and death.

Carboplatin and Paclitaxel Chemoradiation for Localized Anal Cancer in Patients Not Eligible for Mitomycin and 5-Fluorouracil.

BACKGROUND: Although squamous cell carcinoma of the anus (SCCA) is a relatively uncommon malignancy in the United States, it continues to increase in incidence. Treatment for locoregional disease includes mitomycin and 5-fluorouracil with radiation. This combination is associated with significant toxicity, limiting its use in patients who are older or have certain comorbidities. Carboplatin and paclitaxel (C/P) is an accepted treatment regimen for metastatic SCCA. We aim to evaluate the efficacy and toxicity of weekly C/P given with radiation for patients unable to receive standard chemoradiation for SCCA. METHODS: From our cancer registry, adult patients who received weekly intravenous C/P concurrent with standard-dose radiation for localized SCCA were included in this study. Clinical response was determined based on the evidence of disease on imaging and/or anoscopy. Toxicities were graded according to the CTCAE v5. RESULTS: Ten patients were included; eight were female, and the median age was 75.5 years (54-87). Six had T2 disease, and four had T3 tumors. Four had node-positive disease. The majority (70%) of patients were dosed at standard C (AUC 2) and P (50 mg/m2), with a limited subset requiring dose reduction for baseline performance status. Patients completed a mean of 78.3% (40-100%) of the intended treatments. A total of 89% of the patients achieved a complete clinical response. With a median follow-up of 25.8 months (3.4-50.4 months), 67% of the patients are alive and without recurrence. Two patients have had local recurrence, and one patient had metastatic progression. The most common toxicities of any grade included leukopenia (100%), anemia (100%), radiation dermatitis (100%), diarrhea (100%), and fatigue (100%). Grade 3 or higher toxicities included neutropenic fever (20%), neutropenia (30%), and anemia (30%). CONCLUSIONS: This study demonstrates promising tolerability and efficacy for weekly C/P chemoradiation for patients with anal cancer unable to receive mitomycin and 5-fluorouracil. This regimen merits further investigation in prospective clinical trials.

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.

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.

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.

Heme oxygenase-1-mediated autophagy protects against hepatocyte cell death and hepatic injury from infection/sepsis in mice.

UNLABELLED: Adaptive responses to sepsis are necessary to prevent organ failure and death. Cellular signaling responses that limit cell death and structural damage allow a cell to withstand insult from sepsis to prevent irreversible organ dysfunction. One such protective pathway to reduce hepatocellular injury is the up-regulation of heme oxygenase-1 (HO-1) signaling. HO-1 is up-regulated in the liver in response to multiple stressors, including sepsis and lipopolysaccharide (LPS), and has been shown to limit cell death. Another recently recognized rudimentary cellular response to injury is autophagy. The aim of these investigations was to test the hypothesis that HO-1 protects against hepatocyte cell death in experimental sepsis in vivo or LPS in vitro via induction of autophagy. These data demonstrate that both HO-1 and autophagy are up-regulated in the liver after cecal ligation and puncture (CLP) in C57BL/6 mice or in primary mouse hepatocytes after treatment with LPS (100 ng/mL). CLP or LPS results in minimal hepatocyte cell death. Pharmacological inhibition of HO-1 activity using tin protoporphyrin or knockdown of HO-1 prevents the induction of autophagic signaling in these models and results in increased hepatocellular injury, apoptosis, and death. Furthermore, inhibition of autophagy using 3-methyladenine or small interfering RNA specific to VPS34, a class III phosphoinositide 3-kinase that is an upstream regulator of autophagy, resulted in hepatocyte apoptosis in vivo or in vitro. LPS induced phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), in part, via HO-dependent signaling. Moreover, inhibition of p38 MAPK prevented CLP- or LPS-induced autophagy. CONCLUSION: Sepsis or LPS-induced autophagy protects against hepatocellular death, in part via an HO-1 p38 MAPK-dependent signaling. Further investigations are needed to elucidate how autophagic signaling prevents apoptosis and cell death.

Novel management strategies in the treatment of severe Clostridium difficile infection.

CDI is increasing in incidence and severity. Clinicians must have a low threshold to consider the diagnosis and to treat patients with the clinical syndrome and risk factors before laboratory confirmation of the diagnosis. In patients who have signs of advanced disease, escalation of care with antimicrobial strategies and multidisciplinary care including surgical consultation is necessary. Furthermore, lowering the threshold for surgery compared with traditional approaches likely results in improved survival. Novel surgical approaches may obviate total abdominal colectomy and the associated immediate and long-term morbidity in this often fragile patient population, thus allowing clinicians to embrace surgical therapy earlier in the course of severe, complicated disease.

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.

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.