Background: Therapies for metastatic colorectal cancer (CRC) are largely chosen without considering inter-patient heterogeneity, leading to unnecessary side effects without clinical benefit for some patients. Patient-derived cancer organoids (PDCOs) faithfully recapitulate the morphology and molecular profiles of the primary tumors from which they are derived, making them attractive preclinical models for predicting response to standard therapies, and thus, for use in drug development assays. Methods: Here, we investigate the hidden subclonal driver mutations in CRC PDCOs and the importance of individual PDCO-level heterogeneity when addressing PDCO treatment responses using changes in diameter and optical redox imaging. PDCO response is then compared to clinical response across a cohort of subjects with CRC receiving chemotherapy and/or chemoradiation. Results: Change in diameter and optical redox imaging are more sensitive to detecting therapeutic response than endpoint and well-level measurements. These measurements accurately reflect clinical responses to chemotherapy and chemoradiation. Conclusions: Overall, these studies demonstrate the importance of PDCO-level methods of PDCO assessment and further establish PDCOs as powerful tools for drug response assessment and developmental therapeutic studies.

PURPOSE:Versican (VCAN) is an immunoregulatory matrix proteoglycan that, when cleaved, releases an immunostimulatory fragment, versikine. In this study, we evaluate the impact of VCAN on immune surveillance and immunotherapy response in a prospective clinical trial. PATIENTS AND METHODS:T-cell function was assayed in the setting of VCAN. Normal and matched tissues were acquired from 243 patients across colorectal cancer stages. A phase Ib clinical trial enrolled 15 subjects with microsatellite stable oligometastatic colorectal cancer and examined the safety and efficacy of the sequential combination of stereotactic body radiotherapy, pembrolizumab, and resection (NCT02837263). The outcomes were correlated with the VCAN status and circulating immune phenotype by single-cell RNA sequencing. RESULTS:VCAN significantly accumulates in 59% of colorectal cancers and is heavily proteolyzed [VCAN proteolytic predominant (VPP)] in 27% (40% of oligometastatic colorectal cancers). VCAN decreased CD8+ T-cell trafficking (P < 0.01) and activated CD8+ T-cell effector function [decreased IL2RA (P < 0.05), PD-1 (P < 0.05), and granzyme B (GZMB; P < 0.001)]. The clinical trial met the primary endpoint with a 1-year recurrence-free survival (RFS) of 60%. Of those enrolled, 40% had the VPP phenotype, which was associated with a trend toward improved RFS (P = 0.053), and all are still alive with a median follow-up of 4.1 years. The VPP phenotype was associated with enhanced circulating CD8+ T-cell effector function at baseline, which was further enhanced with study treatment. CONCLUSIONS:VCAN limits T-cell trafficking and effector function, and its proteolysis correlates with an effector phenotype of circulating CD8+ T cells, greater tumor-infiltrating lymphocytes, and improved clinical outcomes with this immunotherapy-based clinical trial treatment.

BACKGROUND:When available, anal cytology is used for initial anal cancer screening, whereas diagnostic histology is typically performed after abnormal cytology or in symptomatic individuals. OBJECTIVE:Analyze index anal evaluations (cytology vs histology) in veterans with HIV. DESIGN:Retrospective cohort study. SETTINGS:One hundred thirty Department of Veterans Affairs medical centers. PATIENTS:Veterans with HIV who received care from 1999 to 2023. MAIN OUTCOME MEASURES:Distribution of index anal evaluations as cytology or histology, annual incidence rates of index evaluations, and regional anal cancer prevalence and screening rates. RESULTS:Among 48,368 veterans with HIV, 7127 (15%) had at least 1 anal evaluation. Index evaluations were cytology in 4477 (63%) and histology in 2650 (37%). The mean annual rate of index anal evaluations was 1.04%, with a relative decrease in 2020 and beyond. Among patients with anal evaluations, the Pacific region had the highest proportion of index cytology (77%), comprising 77% of evaluations, and the Continental region had the lowest (37%, p < 0.001). Conversely, the lowest and highest rates of anal cancer were observed in the Pacific and Continental regions, respectively (4.7% vs 9.4%, p < 0.001). However, this trend did not translate into differences in anal cancer rates between the 2 regions when comparing all veterans with HIV (1.01% vs 0.90%, p = 0.52). LIMITATIONS:Retrospective data. CONCLUSIONS:In this national analysis of veterans with HIV, only 15% had an anal evaluation, despite recommendations for annual screening. The majority of index evaluations were screening assessments with cytology (63%). There was a relative decrease in index evaluations after 2020. Regional screening differences were observed among patients with anal evaluations. Higher anal cancer detection was seen in the region with the lowest screening and lower detection in the region with the highest screening. Interestingly, this correlation did not persist when examining the overall regional populations of veterans with HIV, including those never evaluated. See Video Abstract. EVALUACIN DE LAS PRCTICAS DE DETECCIN DEL CNCER ANAL EN UNA COHORTE NACIONAL DE VETERANOS CON VIH:ANTECEDENTES:Cuando está disponible, la citología anal se utiliza para el cribado inicial del cáncer anal, mientras que la histología diagnóstica se suele realizar tras una citología anómala o en personas sintomáticas.OBJETIVO:Analizar las evaluaciones anales índice (citología frente a histología) en veteranos con VIH.DISEÑO:Estudio de cohorte retrospectivo.ENTORNO:Ciento treinta centros médicos del Departamento de Asuntos de Veteranos.PACIENTES:Veteranos con VIH que recibieron atención entre 1999 y 2023.PRINCIPALES RESULTADOS Y MEDIDAS:Distribución de las evaluaciones anales índice como citología o histología, tasas de incidencia anual de las evaluaciones índice y prevalencia regional del cáncer anal y tasas de detección.RESULTADOS:Entre los 48 368 veteranos con VIH, 7127 (15 %) se sometieron al menos a una evaluación anal. Las evaluaciones índice fueron citológicas en 4477 (63 %) y histológicas en 2650 (37 %). La tasa media anual de evaluaciones anales índice fue del 1,04 %, con una disminución relativa a partir de 2020. Entre los pacientes con evaluaciones anales, la región del Pacífico tuvo la mayor proporción de citología índice (77 %), que comprendió el 77 % de las evaluaciones, y la región continental tuvo la más baja (37 %, p < 0,001). Por el contrario, las tasas más bajas y más altas de cáncer anal se observaron en las regiones del Pacífico y continental, respectivamente (4,7 % frente a 9,4 %, p < 0,001). Sin embargo, esta tendencia no se tradujo en diferencias en las tasas de cáncer anal entre las dos regiones al comparar todos los veteranos con VIH (1,01 % frente a 0,90 %, p = 0,52).LIMITACIONES:Datos retrospectivos.CONCLUSIONES:En este análisis nacional de veteranos con VIH, el 15 % se sometió a una evaluación anal, a pesar de las recomendaciones de realizar pruebas de detección anuales. La mayoría de las evaluaciones índice fueron pruebas de detección con citología (63 %). Se observó una disminución relativa de las evaluaciones índice después de 2020. Se observaron diferencias regionales en las pruebas de detección entre los pacientes con evaluaciones anales. Se observó una mayor detección de cáncer anal en la región con menor tasa de detección y una menor detección en la región con mayor tasa de detección. Curiosamente, esta correlación no se mantuvo al examinar la población regional total de veteranos con VIH, incluidos aquellos que nunca fueron evaluados. (AI-generated translation ).

Vulvar squamous cell carcinoma (VSCC) is a rare gynecological cancer with a projection of a 35% increase in the next 20 years in North America. HPV is a known risk factor in VSCC, but HPV+ and HPV- subtypes of VSCC have previously been reported to be biologically but not clinically distinct. In addition, VSCC could be categorized by HPV and TP53 status with different prognoses. However, the VSCC tumor microenvironment (TME) and its contributions to patient clinical outcomes are not well understood. Here, we use single-cell and spatial omics to interrogate VSCC TME transcriptional and cellular heterogeneity as a foundation for understanding the tumor, stromal, and immune cell compartments of HPV+ and HPV-VSCC. Our integrated bioinformatics analysis of 8 scRNA-seq samples (5 HPV+ and 3 HPV-) from vulvar cancer patients identified diverse stromal and immune compartments with immunosuppressive features (high presence of Tregs, pro-tumoral macrophages and myofibroblasts) presenting “immunologically cold” tumor phenotypes. In addition, we identified HPV+ epithelial cells and analyzed HPV-associated cell-cell interactions. Furthermore, we used scRNA-Seq gene signature to deconvolute a bulk 800-gene dataset from an independent patient cohort of 48 samples with different HPV subtypes, including integrated and beta-HPV, to identify TME features associated with different HPV risk subtypes. Bioinformatics deconvolution results showed a new way to subtype VSCC, including a subset of “immunologically hot tumors” (increased CD8 T cells and dendritic cells) with high expression of known and emerging immunotherapy response markers (PD-L1 high, CXCL9 high). Lastly, we integrate scRNA-Seq with patient-matched spatial transcriptomics to identify cellular neighborhood and Ligand-Receptor interactions that may be associated with HPV, subtypes, and clinical outcomes. This study reports a comprehensive single-cell and spatial VSCC TME atlas, highlighting the immunosuppressive features of the TME, differences between HPV+ and HPV- samples, and identification of a subset of patients with “immunologically hot” tumors that may respond favorably to immunotherapy. These findings provide a foundation and resource to further study VSCC TME in both HPV+ and HPV- subtypes. Future studies include validating and studying potential biomarkers of clinical outcomes and immunotherapeutic targets of VSCC treatment. Athena Golfinos-Owens, Taja Lozar, Evie Carchman, Paul F. Lambert, Megan Fitzpatrick, Huy Q. Dinh. Single cell and spatial analysis unravels the immunosuppressive tumor microenvironment, HPV-associated features, and potential immunotherapy response in vulvar squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5265.

Introduction: Pilonidal disease is a chronic skin disorder of the gluteal cleft in adolescents and young adults. No treatment is universally effective, and the heterogeneity of the disease and variability in treatment response frustrates patients and clinicians alike. Surgical treatment strategies focus on the removal of the sinus tracts. Post-operative laser hair removal (LHR) has demonstrated promise to reduce disease recurrence following surgery. LHR has yet to be investigated as a primary treatment strategy and may offer additional benefits. Methods: A single-center prospective pilot study investigated laser hair removal as the primary treatment for moderate to severe pilonidal disease. Patients ages 13-35 with moderate to severe disease who were referred for surgical excision were recruited. Participants underwent 3-8 treatment sessions with the longpulsed-Alexandrite (755 nm) laser by a dermatologist until hair removal endpoints were met. Patients with persistent symptoms after LHR underwent subsequent excision. Patients were subsequently followed in the pediatric surgery clinic at 6, 9,12 and 18 months following LHR to evaluate for disease recurrence. Primary outcomes included resolution rates without surgical intervention and recurrence rates following surgical resection. Secondary outcomes included the number of episodes of infection and impact on quality of life, as assessed by the Dermatology Life Quality Index (DLQI) in patients >= 16 years of age, and the Children's Dermatology Life Quality Index (CDLQI) in patients <16 years of age. Results: Twenty-two patients were enrolled, of which 18 were initiated and 15 completed the LHR sessions in the study, designed to the endpoint of the absence of terminal hair in the gluteal cleft. One patient withdrew prior to completion of the LHR sessions, and two withdrew from the study due to the COVID-19 pandemic. Of the 15 patients who completed the LHR sessions, all demonstrated significant improvement in hair follicle density in the treatment area, with no adverse events. Median number of laser treatments was 6. Six of 15 (40 %) who completed laser treatments had resolution without surgical intervention. Nine patients underwent surgery, of which 6 (67 %) resolved after one surgery, with 18 months of follow up to evaluate for recurrence. Quality of life scores improved after laser treatments (DLQI mean change -4.6, and Children's DLQI mean change -6.0) Conclusion: Laser hair removal was well tolerated, without adverse events and with improved quality of life, in a patient population with moderate to severe pilonidal disease. Nearly half of patients had disease resolution without the need for surgical intervention. These findings underscore the potential for laser hair removal to improve disease outcomes in pilonidal disease, reducing the need for surgical intervention. Based on these results, laser hair removal might provide an effective primary treatment strategy for some patients, and could improve outcomes for those who require surgical intervention. Further research is needed to determine which patients would most likely benefit from this treatment strategy. Level of evidence: Level III. (c) 2025 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Motivation:Cyclic immunofluorescence (IF) techniques enable deep phenotyping of cells and help quantify tissue organization at high resolution. Due to its high dimensionality, workflows typically rely on unsupervised clustering, followed by cell type annotation at a cluster level for cell type assignment. Most of these methods use marker expression averages that lack a statistical evaluation of cell type annotations, which can result in misclassification. Here, we propose a strategy through an end-to-end pipeline using a semi-supervised, random forest approach to predict cell type annotations. Results:Our method includes cluster-based sampling for training data, cell type prediction, and downstream visualization for interpretability of cell annotation that ultimately improves classification results. We show that our workflow can annotate cells more accurately compared to representative deep learning and probabilistic methods, with a training set <5% of the total number of cells tested. In addition, our pipeline outputs cell type probabilities and model performance metrics for users to decide if it could boost their existing clustering-based workflow results for complex IF data. Availability and implementation:Fluoro-forest is freely available on GitHub under an MIT license (https://github.com/Josh-Brand/Fluoro-forest).

Tumor heterogeneity is predicted to confer inferior clinical outcomes with precision-based strategies, however, modeling heterogeneity in a manner that still represents the tumor of origin remains a formidable challenge. Sequencing technologies are limited in their ability to identify rare subclonal populations and predict response to treatments for patients. Patient-derived organotypic cultures have significantly improved the modeling of cancer biology by faithfully representing the molecular features of primary malignant tissues. Patient-derived cancer organoid (PCO) cultures contain subclonal populations with the potential to recapitulate heterogeneity, although treatment response assessments commonly ignore diversity in the molecular profile or treatment response. Here, we demonstrate the advantage of evaluating individual PCO heterogeneity to enhance the sensitivity of these assays for predicting clinical response. Additionally, organoid subcultures identify subclonal populations with altered treatment response. Finally, dose escalation studies of PCOs to targeted anti-EGFR therapy are utilized which reveal divergent pathway expression when compared to pretreatment cultures. Overall, these studies demonstrate the importance of population-based organoid response assessments, the use of PCOs to identify molecular heterogeneity not observed with bulk tumor sequencing, and PCO heterogeneity for understanding therapeutic resistance mechanisms.

Introduction: The incidence of anal cancer is increasing despite screening and treatment options for anal dysplasia, the precursor to anal cancer. Once anal dysplasia is identified, predicting which patients are at the highest risk of progressing to anal cancer remains challenging, as there are no molecular biomarkers for risk stratification. The most common mutation in anal cancer affects the catalytic subunit of Phosphatidylinositol (3, 4, 5)-tri-sphosphate Kinase (PI3K). We sought to determine if PIK3CA mutations are detectable in precancerous anal lesions. Methods: DNA was extracted from formalin-fixed, paraffin-embedded anal tissue slides. Digital polymerase chain reaction was performed to test each sample for the presence or absence of three of the most common PIK3CA mutations: E545 K (c.1633 G > A), H1047 R (c.3140 A > G), and H1047 L (c.3140 A > T). Mutation data, histology, and demographic data were compared. Results: We analyzed 124 tissue samples from 68 unique patients across the spectrum of anal disease. Forty of these samples were E545 K positive, three were H1047 R positive, and two were H1047 L positive. PIK3CA mutations were detected in 8/42 (19%) low-grade dysplasia samples, 14/45 (31%) high-grade dysplasia samples, and 20/37 (54%) cancer samples. The presence of a mutation was associated with higher grade of disease on per-sample analysis (P = 0.004). Conclusions: PIK3CA mutations can be detected in anal tissue samples across the spectrum of carcinogenesis with increasing incidence with higher grade of disease. Our results warrant further evaluation of PIK3CA mutations as a biomarker for identifying patients with anal dysplasia at highest risk of progression to anal cancer.

Despite the availability of several human papillomavirus (HPV) vaccines, the incidence of HPV-associated anal cancer is growing at a rate of 2.2% each year. As shown in results from the recent Phase III ANCHOR study, the treatment of high-grade anal lesions in people living with HIV (PLWH) can significantly reduce rates of anal cancer development compared to active surveillance alone. As a result, screening programs to identify and treat patients with anal precancers are recommended by recent guidelines. Intense resources are needed to perform screening tests and follow-up abnormal results. The lack of effective and well-tolerated therapies, the lack of understanding regarding therapeutic targets, the paucity of preclinical models to test therapies, and the lack of biomarkers to determine which patients will develop cancer or respond to therapies are the issues that need to be addressed. We provide an overview of cutting-edge research and propose additional research that is needed to help move the field of anal cancer prevention forward. This review highlights the most significant current areas of research, as defined by the authors, and is by no means comprehensive of all anal dysplasia/cancer research.

BACKGROUND:Anal squamous intraepithelial lesions are identifiable and treatable precancerous lesions that lack defined risk factors determining screening necessity. OBJECTIVE:Assess the prevalence and risk factors associated with low- and high-grade anal squamous intraepithelial lesions and anal squamous-cell carcinoma. DESIGN:Retrospective cohort analysis of veterans with HIV between 1999 and 2023. SETTINGS:National multicenter study of the Department of Veterans Affairs. PATIENTS:Veterans with HIV who had >1 year of follow-up and no anal squamous intraepithelial lesions or anal cancer diagnosis before the study period. MAIN OUTCOME MEASURES:Primary outcomes include the prevalence, disease-free survival rates, and HRs associated with risk factors for developing anal squamous intraepithelial lesions and/or anal cancer. RESULTS:A total of 48,368 patients were analyzed. The mean age of patients at study initiation was 47.8 years, with a mean follow-up of 12.3 years. Seven thousand five hundred seventy-two patients (16%) had at least 1 anal cytopathology or histopathology result. The prevalence of anal disease was recorded for low-grade disease (n = 1513; 3.1%), high-grade disease (n = 1484; 3.1%), and cancer (n = 664; 1.4%). Mean (SD) times to first incident low-grade disease, high-grade disease, and cancer were 8.5 (6.0), 9.1 (6.0), and 9.7 (6.2) years, respectively. Five-year, 10-year, and 20-year disease-free survival rates for the development of low-grade disease, high-grade disease, or cancer were 97.5%, 94.5%, and 88.4%, respectively. Cox regression modeling demonstrated that CD4/CD8 ratios of <0.5 were associated with an increased risk of anal cancer (HR, 3.93; 95% CI, 3.33-4.63; p < 0.001). LIMITATIONS:Retrospective study that focused almost exclusively on male US veterans. Results might not apply to non-male, non-US populations. CONCLUSIONS:National analysis of more than 48,000 veterans with HIV demonstrates that 16% had anal cytopathology or histopathology results with an anal cancer prevalence of 1.4%. CD4/CD8 ratios of <0.5 correlate strongly with the severity of anal disease and can help identify patients at the highest risk for anal cancer to prioritize screening efforts. See Video Abstract. ANLISIS NACIONAL DE MS DE VETERANOS CON VIH DEMUESTRA QUE LA RELACIN CD/CD ES UN MARCADOR DE RIESGO DE LESIONES INTRAEPITELIALES ANALES Y CNCER ANAL:ANTECEDENTES:Las lesiones intraepiteliales escamosas anales son lesiones precancerosas identificables y tratables que carecen de factores de riesgo definidos que determinen la necesidad de detección.OBJETIVO:Evaluar la prevalencia y los factores de riesgo asociados con las lesiones intraepiteliales escamosas anales de grado bajo y alto y el carcinoma de células escamosas anal.DISEÑO:Análisis de cohorte retrospectivo de veteranos con VIH entre 1999 y 2023.ESTABLECIMIENTO:Estudio multicéntrico nacional del Departamento de Asuntos de Veteranos.PACIENTES:Veteranos con VIH que tuvieron >1 año de seguimiento y sin lesiones intraepiteliales escamosas anales ni diagnóstico de cáncer anal antes del período de estudio.PRINCIPALES RESULTADOS Y MEDIDAS:Los resultados primarios incluyen la prevalencia, las tasas de supervivencia libre de enfermedad y los cocientes de riesgo asociados con los factores de riesgo para desarrollar lesiones intraepiteliales escamosas anales y/o cáncer anal.RESULTADOS:Se analizaron 48.368 pacientes. La edad promedio de los pacientes al inicio del estudio fue de 47,8 años con un seguimiento medio de 12,3 años. 7.572 (16%) pacientes tuvieron al menos un resultado de citopatología o histopatología anal. Se registró la prevalencia de enfermedad anal para enfermedad de bajo grado (n = 1.513, 3,1%), enfermedad de alto grado (n = 1.484, 3,1%) y cáncer (n = 664, 1,4%). Los tiempos medios hasta el primer incidente de enfermedad de bajo grado, enfermedad de alto grado y cáncer fueron 8,5 (DE = 6,0), 9,1 (DE = 6,0) y 9,7 (DE = 6,2) años, respectivamente. Las tasas de supervivencia libre de enfermedad a 5 años, 10 años y 20 años para el desarrollo de enfermedad de bajo grado, enfermedad de alto grado o cáncer fueron 97,5%, 94,5% y 88,4%, respectivamente. El modelo de regresión de Cox demostró que los índices CD4/CD8 <0,5 se asociaban con un mayor riesgo de cáncer anal (HR: 3,93, IC del 95 %: 3,33-4,63, p < 0,001).LIMITACIONES:Estudio retrospectivo que se centra casi exclusivamente en veteranos estadounidenses de sexo masculino. Los resultados podrían no aplicarse a poblaciones no masculinas ni estadounidenses.CONCLUSIONES:El análisis nacional de más de 48 000 veteranos con VIH demuestra que el 16 % tenía resultados de citopatología o histopatología anal con una prevalencia de cáncer anal del 1,4 %. Los índices CD4/CD8 <0,5 se correlacionan fuertemente con la gravedad de la enfermedad anal y pueden ayudar a identificar a los pacientes con mayor riesgo de cáncer anal para priorizar los esfuerzos de detección. (Traducción-Dr Yolanda Colorado ).

Time is a precious commodity for those conducting basic science research. For surgeon-scientists, effective time management is an important skill to master. This includes detailed, advanced planning of experiments for the day, week, month, and beyond. Efficient collection of data will not only provide preliminary data for grant proposals, but will allow for timely publication of results, proper utilization of personnel and funds, fulfillment of commitments, and the establishment of balance—to the extent possible—between work and life outside the laboratory. Ineffective time management can delay your academic advancement, increase personal stress, and decrease personal satisfaction. Instead of trying to create more time in a day, or more days in a week, effective time management allows us to use our time wisely.

This study aims to investigate the phenomenon of high-amplitude pouch contractile waves and their impact on functional results in patients undergoing ileal pouch-anal anastomosis following total proctocolectomy for ulcerative colitis. This is an observational cohort study evaluating pouch manometric data at an early (study 1, < 6 months s/p ileostomy closure) and delayed (study 2, > 5 months after study 1) time point. High-amplitude contractions were defined as peaks ≥ 20 mmHg over baseline. Pouch functional measures and quality of life outcomes were correlated with contractile amplitude and frequency. Thirty-three patients were included in this study. Contractile frequency decreased from study 1 to study 2 (0.14 vs. 0.07 contractions/min). Peristaltic contractility was absent in 18/33 patients (55

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.

Patients with immunodeficiencies and older age are at an increased risk of anal cancer. Transgenic K14E6/E7 mice with established high-grade anal dysplasia were treated topically at the anus with the protease inhibitor saquinavir (SQV) in the setting of CD4+ T-cell depletion to mimic immunodeficiency. To ensure tumor development, specific groups were treated with a topical carcinogen (7,12-Dimethylbenz[a]anthracene (DMBA)). The treatment groups included the vehicle (control), DMBA only, topical SQV, and topical SQV with DMBA, as well as the same four groups with CD4 depletion. The mice were monitored weekly for tumor development. Upon reaching 20 weeks of treatment, the mice were sacrificed, and their anal tissue was harvested for histological analysis. None of the mice in the SQV or control groups developed overt anal tumors, except three mice that were CD4-depleted. The CD4-depleted mice treated with DMBA had significantly increased tumor-free survival and overall survival as well as decreased tumor-volume growth over time when treated with SQV. These data suggest that topical SQV, in the setting of CD4 depletion and high-grade anal dysplasia, can increase tumor-free and overall survival; thus, it may represent a viable topical therapy to decrease the risk of progression of anal dysplasia to anal cancer.

The pathophysiology of the development of anal cancer is thought to be linked to chronic inflammation, a possible consequence of infections with human papillomavirus (HPV) or HIV, or inflammation from inflammatory bowel disease. Anal HPV-induced carcinogenesis bears similarities to its cervical counterpart via viral integration into the host genome and the development of precursor lesions termed anal intraepithelial neoplasia. HPV-16 and -18 are the most common HPV genotypes associated with anal cancer. Other risk factors for the development of anal cancer include chronic immunosuppression, sexual activity and sexually transmitted diseases, female gender, history of anogenital dysplasia, and smoking.

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. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background The over-expression of stress keratin 17 (K17) has been identified as an immune evasion mechanism in a human papillomavirus (HPV) infection mouse model.1 K17 protein expression has also shown to be a prognostic factor in several HPV-associated cancer types, including head and neck (HNC) and cervical cancer.2 3 Our studies suggest K17 is inversely correlated with CD8 T cell infiltration and response to immune check-point blockade (ICB) in HNC.4 We investigated the expression of K17 and markers of immune activation and response to ICB in anogenital cancers. Methods Pre-ICB archival FFPE tissue specimens from anogenital cancer patients undergoing ICB-based therapy at the University of Wisconsin-Madison (UW-Madison, n=15) and University of Alabama-Birmingham (n=13) were stained by immunohistochemistry using a validated K17 monoclonal antibody (Abcam, ab109725). Cases were categorized into K17high vs. K17low, as previously described.4 Study endpoints were progression-free survival (PFS), time to treatment failure (TTF) and overall survival (OS). A tissue microarray (TMA) consisting of archival pretreatment tissue samples from 7 ICB-treated anogenital cancer patients from UW-Madison was subject to multiplex single-cell immunephenotyping on the Akoya Biosciences PhenoCycler-Fusion using a 30-plex antibody panel (table 1) which included a custom K17 antibody compatible with the Phenocycler platform requirements (Novusbio, NBP2–47684). Analysis was performed inQuPath v.0.4.3 using the StarDist (arXiv:1806.03535) nuclear segmentation algorithm and phenotyping using artificial neural network training. Correlations between the expression of markers and clinical outcomes were assessed using the Spearman's coefficient, independent t-test and log rank test. Results Altogether, 28 patients were included in this study (table 2). Fifteen tumors (53.6%) had K17high expression, and 13 tumors (46.4%) had K17low expression (figure 1). K17 status was significantly correlated with TTF (p=0.03), but not PFS or OS (figure 2A). Among patients receiving pembrolizumab-based therapy (n=21), there were 12 (57.1%) K17high vs. 9 (42.9%) K17low tumors. K17 status was again associated with TTF (p=0.007), but not PFS or OS (figure 2B). Altogether, single cell immunephenotyping data from 11 TMA cores from 7 patients (table 3) revealed OS at 6 months was significantly correlated with K17 expression (K17+PanCK+/PanCK+, p=0.032), CD68 (CD68+PanCK-/PanCK-, p=0.016) and PD-L1 on tumor cells (PDL1+PanCK+/all cells, p<0.001), (figure 3). There was no correlation between K17 and individual markers. Conclusions Our findings suggest an inverse trend between K17 expression and clinical outcomes, pending further validation in an expanded patient cohort. References W Wang, et al. 'Stress keratin 17 enhances papillomavirus infection-induced disease by downregulating T cell recruitment,' PLoS Pathog, Jan. 2020;16(1):e1008206, doi: 10.1371/journal.ppat.1008206. L F Escobar-Hoyos, et al. 'Keratin 17 in premalignant and malignant squamous lesions of the cervix: Proteomic discovery and immunohistochemical validation as a diagnostic and prognostic biomarker,' Modern Pathology, 2014;27(4):621–630, doi: 10.1038/modpathol.2013.166. E Regenbogen, et al. 'Elevated expression of keratin 17 in oropharyngeal squamous cell carcinoma is associated with decreased survival,' Head and Neck, 2018;40(8):1788–1798, doi: 10.1002/hed.25164. L P Wang, Lozar T, Golfinos AE, Lee D, Gronski E, Ward-Shaw E, Hayes M, Bruce JY, Kimple RJ, Hu R, Harari PM, Xu J, Keske A, Sondel PM, Fitzpatrick MB, Dinh HQ, 'Stress Keratin 17 Expression in Head and Neck Cancer Contributes to Immune Evasion and Resistance to Immune-Checkpoint Blockade,' Clinical Cancer Research, 2022;28(13):2953–2968. Ethics Approval This study was approved by the Institutional Review Boards at UW-Madison (IRB 2018–1510, subproject 2021–012) and University of Alabama-Birmingham (IRB-300007835).