Sex-dependent development of Kras-induced anal squamous cell carcinoma in mice.

Anal squamous cell carcinoma (SCC) will be diagnosed in an estimated 9,080 adults in the United States this year, and rates have been rising over the last several decades. Most people that develop anal SCC have associated human papillomavirus (HPV) infection (~85-95%), with approximately 5-15% of anal SCC cases occurring in HPV-negative patients from unknown etiology. This study identified and characterized the Kras-driven, female sex hormone-dependent development of anal squamous cell carcinoma (SCC) in the LSL-KrasG12D; Pdx1-Cre (KC) mouse model that is not dependent on papillomavirus infection. One hundred percent of female KC mice develop anal SCC, while no male KC mice develop tumors. Both male and female KC anal tissue express Pdx1 and Cre-recombinase mRNA, and the activated mutant KrasG12D gene. Although the driver gene mutation KrasG12D is present in anus of both sexes, only female KC mice develop Kras-mutant induced anal SCC. To understand the sex-dependent differences, KC male mice were castrated and KC female mice were ovariectomized. Castrated KC males displayed an unchanged phenotype with no anal tumor formation. In contrast, ovariectomized KC females demonstrated a marked reduction in anal SCC development, with only 15% developing anal SCC. Finally, exogenous administration of estrogen rescued the tumor development in ovariectomized KC female mice and induced tumor development in castrated KC males. These results confirm that the anal SCC is estrogen mediated. The delineation of the role of female sex hormones in mediating mutant Kras to drive anal SCC pathogenesis highlights a subtype of anal SCC that is independent of papillomavirus infection. These findings may have clinical applicability for the papillomavirus-negative subset of anal SCC patients that typically respond poorly to standard of care chemoradiation.

Characterisation of anal intraepithelial neoplasia and anal cancer in HIV-positive men by immunohistochemical markers p16, Ki-67, HPV-E4 and DNA methylation markers.

Human papillomavirus (HPV)-induced anal intraepithelial neoplasia (AIN, graded 1-3) is highly prevalent in HIV-positive (HIV+) men who have sex with men (MSM), but only a minority of lesions progresses to cancer. Our study aimed to characterise comprehensively anal tissue samples from a cross-sectional series (n = 104) of HIV+ MSM and longitudinal series (n = 40) of AIN2/3 progressing to cancer using different biomarkers. The cross-sectional series consisted of 8 normal, 26 AIN1, 45 AIN2, 15 AIN3 and 10 anal squamous cell carcinoma. Tissue sections were immunohistochemically (IHC) stained for p16 (viral transformation marker), Ki-67 (cellular proliferation marker) and HPV-E4 (viral production marker). We evaluated the expression of IHC markers and compared it with DNA methylation, a marker for malignant transformation. E4 positivity decreased, whereas p16 and Ki-67 scores and methylation marker positivity increased (P values < .001) with increasing severity of anal lesions. Within AIN2, a heterogeneous biomarker pattern was observed concerning E4, p16 and methylation status, reflecting the biological heterogeneity of these lesions. In the longitudinal series, all AIN2/3 and carcinomas showed high p16 and Ki-67 expression, strong methylation positivity and occasional E4 positivity. We earlier showed that high methylation levels are associated with progression to cancer. The observed E4 expression in some AIN2/3 during the course of progression to cancer and absence of E4 in a considerable number of AIN1 lesions make the potential clinical significance of E4 expression difficult to interpret. Our data show that IHC biomarkers can help to characterise AIN; however, their prognostic value for cancer risk stratification, next to objective methylation analysis, appears to be limited.

PD-L1 expression in anogenital and oropharyngeal squamous cell carcinomas associated with different clinicopathological features, HPV status and prognosis: a meta-analysis.

BACKGROUND: Little research has been done on clinicopathological characteristics and human papillomavirus (HPV) status of anogenital and oropharyngeal squamous cell carcinomas (SCC) with a strong expression of programmed death ligand 1 (PD-L1) in tumor cells. Therefore, we conducted this meta-analysis. METHODS: We performed a comprehensive research in PubMed, Embase and Cochrane databases up to 30 September 2020. The effect size was hazard ratio (HR) with 95% confidence interval (CI) for overall survival (OS), cancer-specific survival (CSS), disease-free survival (DFS). The pooled odds ratio (OR) with 95% CI were used to assess the association between PD-L1 expression and clinicopathological features along with HPV status. RESULTS: A total of 2003 cases (944 anogenital and 1059 oropharynx SCC patients) were included. High PD-L1 expression in anogenital SCC cases were associated with advanced age (OR = 1.63, 95% CI: 1.04-2.58) and HPV negativity (OR = 0.47, 95% CI: 0.31-0.71). Besides, PD-L1 positive anogenital SCC cases held a significantly declined OS (HR = 2.18, 95% CI: 1.37-3.47) and CSS (HR = 2.45, 95% CI: 1.30-4.65). For oropharynx SCC, PD-L1 was more frequent in younger and HPV positive patients (OR = 0.60, 95% CI: 0.37-0.98; OR = 3.01, 95% CI: 1.78-5.09) and PD-L1 expression was relevant to better OS and DFS (HR = 0.76, 95% CI: 0.60-0.97; HR = 0.50, 95% CI: 0.33-0.75). CONCLUSIONS: The meta-analysis demonstrated that in anogenital SCC, PD-L1 positivity had to do with a worse outcome, which might attribute to advanced age, higher tumor grade, lymph node metastasis and HPV negativity, while in oropharynx cancer, PD-L1 expression was related to better prognosis for the reason that PD-L1 was less frequent in the aged and negative HPV status.

Neoadjuvant PD-1 Blockade Combined With Chemotherapy Followed by Concurrent Immunoradiotherapy in Locally Advanced Anal Canal Squamous Cell Carcinoma Patients: Antitumor Efficacy, Safety and Biomarker Analysis.

Background: Anal canal squamous cell carcinoma (ACSCC) is an exceedingly rare malignant neoplasm with challenges in sphincter preservation, treatment toxicities and long-term survival. Little is known concerning the activity of PD-1 antibodies in locally advanced ACSCC. This study reports on the efficacy and toxicities of a neoadjuvant PD-1 blockade combined with chemotherapy followed by concurrent immunoradiotherapy in ACSCC patients, and describes biomarkers expression and mutation signatures. Methods: In this cohort study, patients were treated as planned, including four cycles of neoadjuvant PD-1 antibody toripalimab combined with docetaxol and cisplatin, followed by radiotherapy and two cycles of concurrent toripalimab. Multiplex immunofluorescence staining (mIHC) with PD-L1, CD8, CD163, Pan-Keratin and DAPI was performed with the pretreatment tumor tissue. Whole exome sequencing was performed for the primary tumor and peripheral blood mononuclear cells. The primary endpoint was the complete clinical response (cCR) rate at 3 months after overall treatment. Acute and late toxicities graded were assessed prospectively. Results: Five female patients with a median age of 50 years old (range, 43-65 years old), finished treatment as planned. One patient had grade 3 immune related dermatitis. Two patients had grade 3 myelosuppression during neoadjuvant treatment. No severe radiation-related toxicities were noted. Four patients with PD-L1 expression >1% achieved a cCR after neoadjuvant treatment. and the other patient with negative PD-L1 expression also achieved a cCR at 3 months after radiotherapy. All the patients were alive and free from disease and had a normal quality of life, with 19.6-24 months follow up. Inconsistent expression of PD-L1 and CD163 was detected in 3 and 5 patients, respectively. TTN, POLE, MGAM2 were the top mutation frequencies, and 80 significant driver genes were identified. Pathway analysis showed enrichment of apoptosis, Rap1, Ras, and pathways in cancer signaling pathways. Eight significantly deleted regions were identified. Conclusions: This small cohort of locally advanced ACSCC patients had quite satisfactory cCR and sphincter preservation rate, after neoadjuvant PD-1 antibody toripalimab combined with chemotherapy followed by concurrent immunoradiotherapy, with mild acute and long-term toxicities.

CD4/CD8 Ratio as a Novel Marker for Increased Risk of High-Grade Anal Dysplasia and Anal Cancer in HIV+ Patients: A Retrospective Cohort Study.

BACKGROUND: People living with HIV are at risk for anal dysplasia/cancer. Screening/surveillance is costly and burdensome, and the frequency is not evidence based. Objective markers of increased risk of anal carcinogenesis are needed to tailor screening/surveillance. Low CD4/CD8 ratio is associated with increased overall cancer risk in people living with HIV but has yet to be examined for quantifying anal cancer risk. OBJECTIVE: We hypothesized that low CD4/CD8 ratios correlate with increased risk for high-grade anal dysplasia and cancer. DESIGN: This is a single-institution, retrospective review of people living with HIV from 2002 to 2018. SETTING: This study was conducted at the University of Wisconsin School of Medicine and Public Health. PATIENTS: Patients with advanced disease (high-grade anal dysplasia and/or anal cancer) were compared with patients with negative anal cytology. MAIN OUTCOME MEASURES: The independent variables were lowest (nadir) CD4/CD8 and CD4/CD8 nearest to screening/diagnosis. Logistic regression modeling was used to estimate the adjusted odds of advanced disease. RESULTS: A total of 377 people living with HIV were examined: 266 with negative cytology and 111 with advanced disease (16 cancer, 95 high-grade anal dysplasia). Mean nadir ratio and mean nearest ratio were lower in patients with advanced disease than in those with negative screening (0.26 vs 0.47 (p < 0.001) and 0.61 vs 0.87 (p < 0.001)). In adjusted analyses, increase in nadir ratio or nearest ratio of 1 unit conferred decreased risk of advanced disease (OR, 0.10; 95% CI, 0.02-0.45; p = 0.002) and (OR, 0.31; 95% CI, 0.12-0.83; p = 0.02). The optimal threshold for using CD4/CD8 ratio as a risk factor for advanced disease was 0.47 for nadir ratio (sensitivity 0.59 and specificity 0.91) and 0.95 for nearest ratio (sensitivity 0.56 and specificity 0.92). LIMITATIONS: This is a retrospective, single-institution study. CONCLUSIONS: Low CD4/CD8 ratio confers additional risk of high-grade anal dysplasia and anal cancer beyond the diagnosis of HIV, even when adjusting for known risks factors of anal cancer. Our data suggest that the CD4/CD8 ratio may be able to help identify people living with HIV who are at higher risk of anal cancer development. See Video Abstract at http://links.lww.com/DCR/B336. LA RELACIÓN CD4 / CD8 COMO UN MARCADOR NOVEDOSO PARA EL AUMENTO DEL RIESGO DE DISPLASIA ANAL DE ALTO GRADO Y CÁNCER ANAL EN PACIENTES VIH+: UN ESTUDIO DE COHORTE RETROSPECTIVO: Las personas que viven con el virus de la inmunodeficiencia humana están en riesgo de displasia / cáncer anal. La detección / vigilancia es costosa, laboriosa y la frecuencia no se basa en evidencias. Se necesitan marcadores objetivos de mayor riesgo de carcinogénesis anal para adaptar la detección / vigilancia. La relación baja de CD4 / CD8 se asocia con un mayor riesgo general de cáncer en personas que viven con el virus de inmunodeficiencia humana, pero aún no se ha examinado para cuantificar el riesgo de cáncer anal.Hicimos la hipotesis de que la relación baja de CD4 / CD8 se correlacionan con un mayor riesgo de displasia anal de alto grado y cáncer.Revisión retrospectiva de una sola institución de personas que viven con el virus de la inmunodeficiencia humana desde 2002 hasta 2018.Facultad de Medicina y Salud Pública de la Universidad de Wisconsin.Los pacientes con enfermedad avanzada (displasia anal de alto grado y / o cáncer anal) se compararon con pacientes con citología anal negativa.Las variables independientes más bajas fueron (nadir) CD4 / CD8 y la relación CD4 / CD8 más cercanas a la detección / diagnóstico. Se utilizó el modelo de regresión logística para estimar las probabilidades ajustadas de enfermedad avanzada.Se examinaron un total de 377 personas que viven con el virus de inmunodeficiencia humana, 266 con citología negativa y 111 con enfermedad avanzada (16 cáncer, 95 displasia anal de alto grado). La tasa nadir y la tasa media más cercana fueron más bajas en pacientes con enfermedad avanzada vs. aquellos con cribado negativo (0.26 v. 0.47 (p <0.001) y 0.61 v. 0.87 (p <0.001), respectivamente. En los análisis ajustados, el aumento en la tasa nadir o la tasa más cercana a una unidad confirió una disminución del riesgo de enfermedad avanzada (OR de 0,10 (IC del 95%: 0,02, 0,45, p = 0,002)) y (OR 0,31 (IC del 95%: 0,12, 0,83, p = 0.02)), respectivamente. El umbral óptimo para usar la relacion CD4 / CD8 como factor de riesgo de enfermedad avanzada fue 0,47 para la tasa nadir (sensibilidad 0,59 y especificidad 0,91) y 0,95 para la tasa más cercana (sensibilidad 0,56 y especificidad 0,92).Este es un estudio retrospectivo de una sola institución.La baja relación CD4 / CD8 confiere un riesgo adicional de displasia anal de alto grado y cáncer anal más allá del diagnóstico del virus de inmunodeficiencia humana, incluso cuando se ajustan los factores de riesgo conocidos de cáncer anal. Nuestros datos sugieren que la relación CD4/CD8 puede ayudar a identificar a las personas que viven con el virus de inmunodeficiencia humana que tienen un mayor riesgo de desarrollar cáncer anal. Consulte Video Resumen en http://links.lww.com/DCR/B336.

Role of 18F-FDG PET-derived parameters for predicting complete response to chemoradiotherapy in squamous cell anal carcinoma.

PURPOSE: We aimed to evaluate the accuracy of metabolic parameters, calculated on pretreatment positron emission computed tomography (PET/CT) with fluorodeoxyglucose (F-FDG), for predicting complete response to chemoradiotherapy (CRT) of patients affected by squamous cell anal carcinoma (SCAC). PATIENTS AND METHODS: Clinical records of 20 patients affected by SCAC and treated with CRT were retrospectively evaluated. F-FDG PET/CT was performed at time 0 (baseline) and time 1 (12 weeks after CRT). The following parameters were extracted from PET at time 0: standardized uptake values (SUVmax and SUVmean), metabolic tumor volume (MTV) and total lesion glycolysis (TLG). Response was assessed according to PET response criteria in solid tumors and classified as complete metabolic response (CMR), partial metabolic response (PMR) and progressive metabolic disease (PMD). Receiver operating characteristic analysis was performed to analyze the predictive value of each PET-derived parameter on CMR. RESULTS: Sixteen patients were finally enrolled. All presented increased F-FDG uptake in the primary tumor and 11 (68.7%) also showed metastatic lymph nodes. At PET/CT performed at time 1, 11 subjects (68.7%) presented CMR, three (18.7%) had PMR and the remaining two (12.5%) showed PMD (i.e. hepatic metastases). Among baseline PET-derived parameters, both MTV and TLG efficiently predicted response to CRT with an area under the curve of 0.9 (cutoff 62.3 cm, sensitivity 80%, specificity 100%, P = <0.0001) and 0.87 (cutoff 654.1 g, sensitivity 80%, specificity 100%, P = 0.004), respectively. CONCLUSION: Among PET-derived parameters, both MTV and TLG presented a high predictive value on subjects' outcome after CRT.

Physiological 18F-FDG uptake in the normal adult anal canal: evaluation by PET/CT.

OBJECTIVE: Despite their benefit for detecting primary tumors, data for normal 18F-fluoro-2-deoxy-D-glucose (FDG) uptake in the anal canal are insufficient. Here we used positron emission tomography-computed tomography (PET/CT) to determine the uptake of FDG in the normal adult anal canal (AC) and to evaluate its clinical significance compared with that of anal cancer. METHODS: We conducted a retrospective study of-PET/CT images in the anal region, of 201 consecutive patients without symptoms or pathology taken from January 2015 to August 2019, after excluding two patients (one each with Crohn's disease and hemorrhoid). These patients were included in the normal group, and data of eight patients with anal cancer were collected from January 2011 to August 2019 for comparison. FDG uptake was quantitatively evaluated (compared with the maximum standardized uptake value [SUVmax] to the SUVmax values of liver and distal rectum) and qualitatively (compared with background) in early and delayed phases. Normal grade 3 uptake was qualitatively defined as FDG uptake higher than the surrounding muscles. RESULTS: In the normal group, mean anal canal SUVmax of early phase was: 2.26 (range 1.00-6.30), and delayed phase: 2.52 (range 1.00-8.80). Their ratios to liver SUVmax were early: 0.74 (range 0.24-2.25), and delayed: 0.81 (range 0.23-2.32); ratios to rectal SUVmax were early: 0.87 (range 0.30-1.89), and delayed: 0.90 (range 0.30-1.27). Qualitatively, 25 patients (15.4%) had normal grade 3 uptake during the early and delayed phases. In contrast, qualitative data showed that all patients with anal cancer exhibited high FDG uptake in the anal canal. The mean early- and delayed-phase values of SUVmax of the anal canal and anal cancer group were 11.09 (range 5.40-17.73) and 14.23 (range 6.70-22.85), respectively. There was a significant difference between the mean-early and -delayed anal SUVmax values of the normal grade 3 and anal cancer groups. Furthermore, the ratios to liver SUVmax were significantly different between the two groups. CONCLUSIONS: PET/CT scans occasionally showed high FDG uptake in the anal canal of healthy adults. Comparing the SUVmax values of liver FDG uptake may help differentiate between normal tissue and anal cancer.

Elevation of Serum CEA in Patients with Squamous Cell Carcinoma of the Anus.

The proportion of anal cancer cases that produce elevated carcinoembryonic antigen (CEA) levels is not well described in the medical literature. In this study, we used electronic health record data from a single urban cancer center to identify patients from 2004-2018 with anal cancer who have also had a pre-initial treatment CEA measurement. We identified 40 patients who met our eligibility criteria. Of those, 11 (27.5%) had an elevated pretreatment CEA. Elevated CEA was not associated with any of the clinical or demographic covariates; however, three out of five patients with a recurrence had an elevated CEA.

A Unique Case of Primary Extranodal Marginal Zone Lymphoma of the Anal Canal.

Marginal zone lymphomas represent approximately 10-12% of all B-cell lymphomas. Extranodal marginal zone lymphomas (EMZL) or mucosa-associated lymphoid tissue (MALT) lymphomas are the most common subtype. Almost half of all MALT lymphomas arise in the gastrointestinal (GI) tract and, while the stomach is the most common site of GI involvement, the small and large intestines can also be involved. Rare cases of MALT lymphoma involving the rectum have been reported; however, to our knowledge, involvement of the anal canal has never been reported in the literature. Here, we describe a unique case of MALT lymphoma of the anal canal. Infectious agents have been implicated in the pathogenesis of MALT lymphomas, possibly through persistent antigenic stimulation of the area; however, in our case no such infection was documented.

Interaction between IGF2-PI3K axis and cancer-associated-fibroblasts promotes anal squamous carcinogenesis.

Anal squamous cell carcinoma (ASCC) is a rare tumour, but its incidence is increasing. Standard chemoradiotherapy fails to cure 20% of patients and no targeted therapy is currently approved for recurrent ASCC. The PI3K/Akt/mTOR pathway is frequently altered in this poorly characterised carcinoma. IGF2 was identified here as a key factor in ASCC oncogenesis, as IGF2 was shown to play a crucial role in the PI3K pathway with frequent (~60%) and mutually exclusive genomic alterations in IGF2, IGF1R, PTEN and PIK3CA genes. We also demonstrated that IGF2 expression is mainly due to cancer-associated fibroblasts and that IGF2 secreted by cancer-associated fibroblasts from ASCC samples promotes proliferation of a human ASCC cell line via IGF2 paracrine signalling. Altogether, these results highlight the key role of the IGF2/PI3K axis, and the major role of cancer-associated fibroblasts in tumour growth via IGF2 secretion, suggesting a major role of IGF2/IGF1R inhibitors in ASCC therapies.

Utility of Stathmin-1 as a Novel Marker in Evaluating Anal Intraepithelial Neoplasia (AIN).

The aim of this study is to determine whether immunohistochemistry for Stathmin-1 enhances diagnostic accuracy of anal dysplasia. The study included 40 biopsies with diagnosis of benign anal transitional zone (n=10), low-grade anal intraepithelial neoplasia (AIN) (AIN1, n=10), and high-grade AIN (AIN2, n=10, AIN3, n=10). The cases were selected to represent classic features. Immunohistochemistry for Stathmin-1, p16, and Ki-67 was performed and assessed for distribution within epithelial thickness. Stathmin-1 was expressed only in the basal layer of benign anal epithelium. Similar pattern of distribution was seen in all low-grade AIN cases (100%). In total, 40% of AIN2 showed Stathmin-1 staining pattern similar to AIN1. The other 60% of cases showed staining extending into the middle third of the epithelial thickness. Of AIN3 cases, 20% showed staining confined to the lower third epithelium, 20% showed staining extending to the middle third, and 60% showed staining extending into the upper third epithelium. The pattern of stain distribution suggested that staining extending above the lower one-third of the epithelial thickness discriminates between low-grade and high-grade AIN. With this cutoff, the sensitivity for the diagnosis of high-grade AIN was 70%, specificity was 100%, positive predictive value equaled 100%, and negative predictive value equaled 77%. P16INK4a showed 100% sensitivity for AIN2 and AIN3, whereas Ki-67 had 100% sensitivity for any AIN grade. In conclusion, Stathmin-1 has excellent specificity for the diagnosis of high-grade AIN; however, Stathmin-1 alone may not be sufficiently sensitive. Use in conjunction with other sensitive markers, such as p16 or Ki-67 may be considered.

Human Papillomavirus (HPV)-associated Lymphoepithelioma-like Carcinoma of the Vagina and Anal Canal: A Rare Variant of Squamous Cell Carcinoma.

Lymphoepithelioma-like carcinoma (LELC) is an uncommon variant of squamous cell carcinoma, which is histologically identical to lymphoepithelial carcinoma of the nasopharynx. LELCs have been reported at a variety of sites, including the stomach, salivary gland, thymus, cervix, endometrium, breast, skin, bladder, and lung. We report 2 LELCs of the vagina and 1 of the anal canal, the first report of LELC at the latter site. All 3 neoplasms were diffusely positive with p16 (block-type immunoreactivity) and the anal canal lesion contained high-risk human papillomavirus type 16; the 2 vaginal neoplasms underwent human papillomavirus testing but were unsuitable for analysis. All cases were Epstein-Barr virus negative. In reporting these cases, we highlight the potential for misdiagnosis and suggest an association with human papillomavirus infection similar to LELCs in the uterine cervix.

Pathology Characterization and Detection of Human Papillomavirus Type 16 in Rectal Squamous Cell Carcinomas.

Rectal squamous cell carcinoma (SCC) is a rare tumor with unresolved etiology. Human immunodeficiency virus-infected individuals and solid organ transplant recipients experience >30-fold and approximately 3-fold elevated rates of rectal SCC, respectively, suggesting immunosuppression plays a role.1 Human immunodeficiency virus-infected homosexual men have >60-fold higher rates of rectal SCC, similar to anal SCC. These patterns, which differ from the more common rectal adenocarcinoma (AdCA), raise the possibility of shared etiology between rectal and anal SCC, with human papillomavirus type 16 (HPV16) being a likely candidate.2.

Beyond p16 immunostaining: an overview of biomarkers in anal squamous intraepithelial lesions.

Histological grading of squamous intraepithelial lesions or intraepithelial neoplasia is fundamental for clinical management and for assessment of the risk of progression. Biomarkers are important for assisting correct grading of these lesions, reducing inter and intraobserver variability and most promising, for prognosis. Although p16 is the most studied biomarker in this setting, there are several other biomarkers that have been studied, reflecting also the need to find a better single or association option that can be more suitable, especially for classification purposes. A PubMed and Embase search was conducted from their inception until April 2018, aiming to identify biomarkers evaluated in histological samples of anal squamous intraepithelial lesions, other than p16. Information on "Ki-67", "ProExTM C", "p53", "human papillomavirus L1 capsid protein", "stathmin-1", "minichromosome maintenance protein", "p21", "proliferating cell nuclear antigen", "histones", "human papillomavirus E4", "chromosomal abnormalities" and "methylation" was collected and reviewed. From these, the most studied biomarker was by far Ki-67. In many cases there were few studies performed for each biomarker, with no clear standardized interpretation of the immunostaining. An increased positive rate with more severe grades of lesions was shown in many cases. Prognostic data are limited and need to be further validated.

Primary anorectal melanoma: clinical, immunohistology and DNA analysis of 43 cases.

Primary melanoma involving the anorectal region is rare, accounting for <1% of all melanomas in most Western countries. It characteristically presents at an advanced clinical stage and is associated with poor clinical outcomes. Preliminary reports suggest that response rates to immunotherapies in patients with advanced stage mucosal melanoma are much lower than in cutaneous (or acral) melanoma patients but reasons for this are unclear. Comprehensive characterisation of the immune microenvironment in anorectal melanoma has not previously been performed. A single-institution cohort of 43 primary anorectal melanoma patients was examined to describe clinicopathological features and characterise the immune microenvironment to provide insights into the behaviour of this rare melanoma subtype. The tumours displayed multiple adverse prognostic attributes including deep thickness (median 11.5 mm), ulceration (81%) and high mitotic rate (median 12/mm2). The median overall survival was 24 months and the median recurrence-free survival was 9 months. Tumour-infiltrating lymphocytes (TILs) were absent or mild in most tumours (75%); PD-L1 positive staining (>1% of tumour cells) was present in 44% of cases, however in 86% of positive tumours the percentage of positive cells was ≤10%. Four tumours underwent whole genome sequencing; no ultraviolet signature was identified, and there was a lower mutational load but higher structural chromosomal variant load compared with cutaneous melanomas. Poor responses of anorectal melanomas to immunotherapy may be caused by lower immunogenicity of these tumours as characterised by low mutation burden (and therefore low neoantigenicity), low TILs infiltrates and low PD-L1 expression. Further investigation is required to determine whether TILs and PD-L1 expression predict response to immunotherapy in patients with mucosal melanoma.

Anorectal Melanoma - a Histopathological Case Report and a Review of the Literature.

Primary melanomas of the anus and rectum are rare neoplasms with aggressive behavior, accounting for 0.1%-4.6% of anal canal tumors. Mucosal melanomas account for approximately 1.2% of all melanomas, of which fewer than 25% are anorectal. Histological evaluation with immunohistochemical stains like HMB-45, S-100, vimentin and Melan A is required for definitive diagnosis. The 5-year survival rate for anorectal melanomas (AM) was reported to be as low as < 20%, in contrast to the value of approximately 80% for cutaneous melanomas. Furthermore, up to 67% of patients are found to have distant metastases at the time of their initial diagnosis with AM. Since the chemotherapy treatment possibilities are limited, patients usually undergo mutation detection tests giving the opportunity of targeted therapy. Herein we report a case of a patient with anorectal melanoma, diagnosed in stage II and the pathomorphological and mutation status finding, together with their correlation to tumor behavior and patient prognosis.

Programmed Cell Death-Ligand 1 (PD-L1) Expression in Anal Cancer.

OBJECTIVE: To evaluate the expression of programmed cell death-ligand 1 (PD-L1) in anal cancer. PATIENTS AND METHODS: In a retrospective cohort analysis, subjects with squamous cell carcinoma of the anal canal were tested for PD-L1 expression, then followed for recurrence and survival. Crude recurrence rates (CRRs), crude mortality rates (CMRs), and crude event rates (CERs) were assessed for PD-L1-dependent differences using Poisson regression. All 3 types of crude rate were expressed as the number that occurred per hundred person-years (hPY) of follow-up. RESULTS: Samples from 41 subjects were evaluated for PD-L1 expression; 23 (56%) were positive. Subjects with PD-L1-expressing versus PD-L1-negative tumors respectively had CRRs of 30.8 versus 12.1 recurrences/hPY (P=0.082), CMRs of 16.7 versus 12.0 deaths/hPY (P=0.47), and CERs of 39.2 versus 16.9 events/hPY (P=0.069). CONCLUSIONS: PD-L1 positivity was associated with worse CRR and CER, and marginally worse CMR. The effect on progression-free and overall survival needs to be validated in a study with a larger sample size.

Prognostic impact of RITA expression in patients with anal squamous cell carcinoma treated with chemoradiotherapy.

BACKGROUND: RBP-J interacting and tubulin-associated protein (RITA) has been identified as a negative regulator of the Notch signalling pathway and its deregulation is involved in the pathogenesis of several tumour entities. RITA's impact on the response of anal squamous cell carcinoma (SCC) to anticancer treatment, however, remains elusive. MATERIALS AND METHODS: In our retrospective study immunohistochemical evaluation of RITA was performed on 140 pre-treatment specimens and was correlated with clinical and histopathologic characteristics and clinical endpoints cumulative incidence of local control (LC), distant recurrence (DC), disease-free survival (DFS) and overall survival (OS). RESULTS: We observed significant inverse correlations between RITA expression and tumour grading, the levels of HPV-16 virus DNA load, CD8 (+) tumour infiltrating lymphocytes and programmed death protein (PD-1) immunostaining. In univariate analyses, elevated levels of RITA expression were predictive for decreased local control (p = 0.001), decreased distant control (p = 0.040), decreased disease free survival (p = 0.001) and overall survival (p < 0.0001), whereas in multivariate analyses RITA expression remained significant for decreased local control (p = 0.009), disease free survival (p = 0.032) and overall survival (p = 0.012). CONCLUSION: These data indicate that elevated levels of pretreatment RITA expression are correlated with unfavourable clinical outcome in anal carcinoma treated with concomitant chemoradiotherapy.

Unsatisfactory exfoliative anal cytology samples, 15-year experience with histologic, cytologic, and molecular follow-up.

BACKGROUND: The incidence of anal carcinoma has risen in recent decades. Exfoliative cytology screening of selected high risk patients is performed in many centers. Unsatisfactory cytology results are frustrating to patients, clinicians, and laboratorians. The aim of this study is to ascertain outcomes of patients with non-diagnostic anal cytology. METHODS: A retrospective review of anal cytology testing performed at the Cleveland Clinic between 01/01/2001 and 12/31/2015 was performed. All cases were received as liquid-based samples and processed as ThinPreps (Hologic, Marlborough, MA). Co-testing for HR-HPV DNA was performed using Hybrid Capture 2® (Qiagen, Germantown, MD) in the majority of patients. RESULTS: Of 1,276 ThinPrep anal cytology samples, 130 (10%) were deemed unsatisfactory. 77% of patients were HIV positive. 85% were males. Of the unsatisfactory cases, 116 (89%) were co-tested for HR-HPV DNA. Of those, 40 patients (34%) had a simultaneous positive HR-HPV DNA. Adequate follow up cytology within a one year and a two year period revealed that 18/130 (14%) and 26/130 (20%) of patients had ASC or SIL respectively. Histologic follow-up within one and two years showed 3 patients (2%) and 8 patients (6%) with HSIL or worse. CONCLUSIONS: High risk patients with unsatisfactory anal cytology are not "negative". At least one-third proved to be concomitantly HR-HPV DNA positive with one-fifth showing subsequent cytologic squamous abnormalities and with more than 5% being diagnosed with a high grade intraepithelial lesion within two years. Prompt repeat cytology and/or HR-HPV DNA is recommended for high risk patients with non-diagnostic cytology.

Role of 18F-FDG PET/CT in Posttreatment Evaluation of Anal Carcinoma.

The aim of this study was to evaluate the relevance of PET/CT and 18F-FDG as a strategy for response evaluation after chemoradiotherapy for anal cancer. For this, the performance of posttreatment 18F-FDG PET/CT, the impact on patient care, and the predictive value of metabolic response were assessed. Methods: This was a retrospective and multicenter analysis of 87 patients treated by chemoradiotherapy for anal squamous cell carcinoma between October 2007 and October 2013. All patients underwent systematic posttreatment 18F-FDG PET/CT and were followed with at least a clinical examination every 4 mo for 2 y and every 6 mo thereafter. Disease progression was confirmed by biopsy for all patients in the case of local recurrence before surgery. Kaplan-Meier and Cox regression models were used to test for associations between metabolic or clinical endpoints and progression-free survival (PFS) or cause-specific survival (CSS). Results: The median follow-up was 25 mo. 18F-FDG PET/CT was performed 1-8 mo (median, 4 mo) after completion of chemoradiotherapy. Overall, 25 patients relapsed and 13 died. The posttherapy 18F-FDG PET/CT did not show any abnormal 18F-FDG uptake (complete metabolic response [CMR]) in 55 patients whereas 32 displayed incomplete response (non-CMR): 15 patients with partial response and 17 with disease progression. The sensitivity of 18F-FDG PET/CT to detect residual tumor tissue was 92% (95% confidence interval [CI], 75%-97%), specificity was 85% (95% CI, 75%-92%), positive predictive value was 72% (95% CI, 61%-90%), and negative predictive value was 96.4% (95% CI, 90%-98.7%). The 2-y PFS was 96% (95% CI, 90-100) for patients with CMR and 28% (95% CI, 14-47) for non-CMR patients (P < 0.0001). The 2-y CSS was 100% for patients with CMR and 59% (95% CI, 42-84) for those without CMR (P < 0.0001). 18F-FDG PET/CT changed patient management in 14 cases (16%), with relevant modifications in 12 (14%). A Cox proportional hazards model of survival outcome indicated that a CMR was the only significant predictor of PFS and CSS (P < 0.0001). Conclusion:18F-FDG PET/CT shows good accuracy in posttreatment evaluation of anal cancer and has a relevant impact on patient management. Moreover, CMR is associated with good survival outcome. Thus, 18F-FDG PET/CT may play a significant role during posttreatment follow-up of anal cancer.