Peritoneal Washings Research Articles

Anti-Tumor Immunogenicity of the Oncolytic Virus CF33-hNIS-antiPDL1 against Ex Vivo Peritoneal Cells from Gastric Cancer Patients.

We studied the immunotherapeutic potential of CF33-hNIS-antiPDL1 oncolytic virus (OV) against gastric cancer with peritoneal metastasis (GCPM). We collected fresh malignant ascites (MA) or peritoneal washings (PW) during routine paracenteses and diagnostic laparoscopies from GC patients (n = 27). Cells were analyzed for cancer cell markers and T cells, or treated with PBS, CF33-GFP, or CF33-hNIS-antiPDL1 (MOI = 3). We analyzed infectivity, replication, cytotoxicity, CD107α upregulation of CD8+ and CD4+ T cells, CD274 (PD-L1) blockade of cancer cells by virus-encoded anti-PD-L1 scFv, and the release of growth factors and cytokines. We observed higher CD45-/large-size cells and lower CD8+ T cell percentages in MA than PW. CD45-/large-size cells were morphologically malignant and expressed CD274 (PD-L1), CD252 (OX40L), and EGFR. CD4+ and CD8+ T cells did not express cell surface exhaustion markers. Virus infection and replication increased cancer cell death at 15 h and 48 h. CF33-hNIS-antiPDL1 treatment produced functional anti-PD-L1 scFv, which blocked surface PD-L1 binding of live cancer cells and increased CD8+CD107α+ and CD4+CD107α+ T cell percentages while decreasing EGF, PDGF, soluble anti-PD-L1, and IL-10. CF33-OVs infect, replicate in, express functional proteins, and kill ex vivo GCPM cells with immune-activating effects. CF33-hNIS-antiPDL1 displays real potential for intraperitoneal GCPM therapy.

Increased susceptibility to encephalitozoonosis associated with mixed Th1/Th2 profile and M1/M2 profile in mice immunosuppressed with cyclophosphamide

Encephalitozoon cuniculi is a unicellular, spore-forming, obligate intracellular eukaryote belonging to the phylum Microsporidia. It is known to infect mainly immunocompromised and immunocompetent mammals, including humans. The parasite–host relationship has been evaluated using both in vitro cell culturing and animal models. For example, Balb/c and C57BL/6 mouse strains have been used interchangeably, although the latter has been considered more susceptible due to the higher fungal load observed after infection. In the present study, we identified the characteristics of the immune response of C57BL/6 mice treated or not with the immunosuppressant cyclophosphamide (Cy) and challenged with E. cuniculi by intraperitoneal route. After 14 days of infection, serum was collected to analyze Th1, Th2, and Th17 cytokine levels. In addition, peritoneal washes were performed, and the spleen sample was collected for immune cell phenotyping, whereas liver, spleen, kidney, lung, intestine, and central nervous system (CNS) samples were collected for histopathological analysis. Although infected mice displayed a reduced absolute number of macrophages, they showed an M1 profile, an elevated number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (interferon [IFN]-γ, tumor necrosis factor [TNF]-α, and interleukin [IL]-2) and Th17. Furthermore, Cy-Infected mice showed a reduced absolute number of macrophages with an M1 profile but a reduced number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th2 (IL-4). This group displayed a higher fungal burden as well and developed more severe encephalitozoonosis, which was associated with a reduced number of T and B lymphocytes and a mixed profile of Th1 and Th2 cytokines.

Development of an Artificial Intelligence Model for Classifying Major Cell Types in Peritoneal Washings from Patients with Ovarian High-Grade Serous Carcinoma

Development of an Artificial Intelligence Model for Classifying Major Cell Types in Peritoneal Washings from Patients with Ovarian High-Grade Serous Carcinoma

Concordance Rate Between Cytology and KRAS Mutational Analysis in Peritoneal Washings of Patients with Pancreatic Ductal Adenocarcinoma

Concordance Rate Between Cytology and KRAS Mutational Analysis in Peritoneal Washings of Patients with Pancreatic Ductal Adenocarcinoma

Cytopathologie des séreuses : la classification internationale : À propos des techniques ancillaires prédictives et du cas particulier des lavages péritonéaux

The International System for serous fluids cytopathology is a cytologic classification which purpose is to establish a consensus on diagnostic terminology. The exponential discovery of prognostic and theranostic molecular alterations in many cancers, particularly in advanced stages, led the authors to describe the indications and the feasibility of these new markers on cytological samples from serous effusions. The various immunocytochemistry techniques, FISH and those testing DNA and RNA are reported in regard to their ability to identify the main targets currently explored in routine practice. The vast majority of these crucial markers can be reliably tested on effusion fluids. The International System for serous effusion fluids also includes a chapter dedicated to peritoneal washings and the application of the classification to this particular type of sample. The objective is to "wash" the peritoneal cavity with a saline solution to unfix cells from the cavity's wall and collect those that have previously naturally detached. This procedure, performed before surgery of tumors and before any manipulation, allows a cytological analysis that specifies the staging of gynecological and non-gynecological tumors and excludes occult malignant cells in the presence of tumors appearing benign.

Current opportunities for combined treatment of gastric cancer patients with peritoneal carcinomatosis

Patients with locally advanced gastric cancer are at high risk of peritoneal dissemination. Poor prognosis after surgical treatment is mostly associated with presence of free tumor cells in peritoneal cavity that were not identified during pre-surgery examination. Staging laparoscopy and peritoneal washing with cytological examination are now routinely used to detect peritoneal carcinomatosis and for an accurate cancer staging.Yet there is no standard treatment for patients with morphologically confirmed peritoneal dissemination of gastric origin. International oncological associations such as NCCN, ESMO and AJCC identify presence of free tumor cells in peritoneal cavity as distant metastasis where palliative chemotherapy is recommended as the only treatment option. Literature review shows that even if complete regression of micrometastases is achieved after chemotherapy alone, survival rate of this group of patients remains poor.Today some authors prove combined treatment strategies for patients with peritoneal micrometastases of gastric origin to be effective. Because of the limited number of patients included in these studies there is no specific combined treatment scheme that can be recommended as a standard protocol of treatment patients with advanced gastric cancer.

Evaluating staging laparoscopy indications for pancreatic cancer based on resectability classification and treatment strategies for patients with positive peritoneal washing cytology.

The prognosis of pancreatic ductal adenocarcinoma (PDAC) in patients with positive peritoneal washing cytology (CY1) is poor. We aimed to evaluate the results of staging laparoscopy (SL) and treatment efficacy in CY1 patients based on a resectability classification. We retrospectively reviewed 250 patients with PDAC who underwent SL before the initial treatment between 2017 and 2023 at the University of Toyama. The breakdown of cases by resectability classification was resectable (R):borderline resectable (BR):unresectable locally advanced (UR-LA) = 131:48:71 cases. The frequency of CY1 increased in proportion to the degree of local progression (R:BR:UR-LA = 20:23:34%), but the frequencies of liver metastasis or peritoneal dissemination were comparable (R:BR:UR-LA = 6.9:6.3:8.5%). Most CY1 patients received gemcitabine along with nab-paclitaxel therapy. The CY-negative conversion rates (R:BR:UR-LA = 70:64:52%) and conversion surgery rates (R:BR:UR-LA = 40:27:9%) were inversely proportional to the degree of local progression.Comparing H0P0CY1 factors for each classification, patients with H0P0CY1 had significantly more pancreatic body or tail carcinoma and tumor size ≥32 mm in R patients, whereas in BR patients, duke pancreatic monoclonal antigen type 2 (DUPAN-2) ≥ 230 U/mL was a significant factor. In contrast, no significant factors were observed in UR-LA patients. The CY1 rates, CY-negative conversion rates, and conversion surgery rates varied according to local progression. In the case of R and BR, SL could be considered in patients with pancreatic body or tail carcinoma, large tumor size, or high DUPAN-2 level. In UR-LA, SL might be considered for all patients.

Outcomes for patients with high-risk endometrial cancer undergoing sentinel lymph node assessment versus full lymphadenectomy

ObjectiveThe objective of this study was to determine the progression free survival (PFS) and overall survival (OS) among patients with high-risk endometrial cancer (EC) who underwent sentinel lymph node (SLN) mapping and dissection compared to patients who underwent pelvic +/− para-aortic lymphadenectomy (LND). MethodsPatients with newly diagnosed high-risk EC were identified. Inclusion criteria included patients who underwent primary surgical management from January 1, 2014 to September 1, 2020 at our institution. Patients were categorized into either the SLN or LND group based on their method of planned lymph node assessment. Patients in the SLN group had dye injected followed by successful bilateral lymph node mapping, retrieval, and processing per our institutional protocol. Clinicopathological and follow-up data were extracted from patient's medical records. The t-test or Mann-Whitney test was used to compare continuous variables and Chi-squared or Fisher's exact test were used for categorical variables. Progression-free survival (PFS) was calculated from the date of initial surgery to the date of progression, death, or last follow-up. Overall survival (OS) was calculated from the date of surgical staging to the date of death or last follow-up. Three-year PFS and OS were calculated using the Kaplan-Meier method, and the log-rank test was used to compare cohorts. Multivariable Cox regression models were used to assess the relationship between nodal assessment cohort and OS/PFS while adjusting for age, adjuvant therapy, and surgical approach. A result was considered statistically significant at the p < 0.05 level of significance and all statistical analysis was done using SAS version 9.4 (SAS Institute, Cary, NC). ResultsOut of 674 patients diagnosed with EC during the study period, 189 were diagnosed with high-risk EC based on our criteria. Forty-six (23.7%) patients underwent SLN assessment and 143 (73.7%) underwent LND. No difference was observed between the two groups in regards to age, histology, stage, body mass index, tumors myometrial invasion, lymphovascular space invasion, or peritoneal washing positivity. Patients in the SLN group underwent robotic-assisted procedures more frequently than those in the LND group (p < 0.0001). The three-year PFS rate was 71.1% (95% CI 51.3–84.0%) in the SLN group and 71.3% (95% CI 62.0–78.6%) in the LND group (p = 0.91). The unadjusted hazard ratio (HR) for recurrence in the SLN versus LND group was 1.11 (95% CI 0.56–2.18; p = 0.77), and after adjusting for age, adjuvant therapy, and surgical approach, the HR for recurrence was 1.04 (95% CI 0.47–2.30, p = 0.91). The three-year OS rate was 81.1% (95% CI 51.1–93.7%) in the SLN group and 95.1% (95% CI 89.4–97.8%) in the LND group (p = 0.009). Although the unadjusted HR for death was 3.74 in the SLN vs LND group (95% CI 1.39–10.09; p = 0.009), when adjusted for age, adjuvant therapy, and surgical approach, it was no longer significant with a HR of 2.90 (95% CI 0.94–8.95, p = 0.06). ConclusionsThere was no difference in three-year PFS in patients diagnosed with high-risk EC who underwent SLN evaluation compared to those who underwent full LND in our cohort. The SLN group did experience shorter unadjusted OS; however, when adjusting for age, adjuvant therapy and surgical approach, there was no difference OS in patients who underwent SLN compared to LND.

Proteomic profiling of peritoneal fluid-derived exosomes as a tool for the identification of prognostic markers in advanced ovarian carcinomas.

e17561 Background: Exosomes have been involved in tumor progression and development of therapeutic resistances in several malignancies. This subtype of extracellular vesicles (EVs) promote angiogenesis, premetastatic niches formation and communication between the tumor and its microenvironment. Ovarian cancer (OC) is a highly lethal tumor that typically spreads within the abdominal cavity rather than through blood vessels. Based on this behavior, we aimed to study the presence of EVs in ascitic fluid of OC patients and explore their potential as predictors of tumor volume and chemoresistance. Methods: Observational prospective study in advanced OC patients who underwent a diagnostic laparoscopy or cytoreductive surgery. Abdominal washings of a cohort of non-cancer patients were collected as controls. All cases provided written consent. Clinical data and tumor genomic alterations (NGS, FoundationOne CDx) were recorded. EVs pellets were collected by ultracentrifugation and profiled by NTA and western blot (EVs markers: CD9, TSG101 and ALIX; non EVs related markers: ALB and APOB). EVs proteins were quantified by BCA assay and profiled by mass spectrometry. Results: We obtained 75 peritoneal fluids from 66 OC patients (median age at diagnosis [range] 62 [26-83] years) and 29 peritoneal washings from controls. Our cohort comprises various histological subtypes (high grade serous, n 55; low grade serous, n 2; endometrioid, n 6; clear cell, n 2 and mucinous, n 1) and tumor stages (I-II, n 7; III; n 39; IV, n 20). Samples were collected either from primary (n 36); interval, (n 25); or relapse surgery (n 14). Pathogenic BRCA events: 15 patients. Mean concentration of exosomes (MCE) was 3.0x106 vs. 1.6x106 particles/ml (p/ml) in cases vs. controls (p &lt; 0.0017). No difference in MCE was observed regarding stages I-II vs. III-IV (3.9x106 vs. 3.3x106 p/ml; p = 0.38). A higher MCE was observed in those samples collected after relapse surgeries compared to those collected from primary (7.2x106 vs. 2.8x106 p/ml; p &lt; 0.0001) and interval surgeries (7.2x106 vs. 3.4x106 p/ml; p &lt; 0.0007). Median exosomal protein concentration (MEPC) in EVs of cancer patients vs. controls was 0.99µg/µl and 0.57µg/µl respectively (p = 0.02). Proteomic characterization by mass spectrometry of EVs cargo has identified 1827 proteins, of which 405 proteins were differentially expressed between cases and controls. We also found 38 differentially expressed proteins comparing platinum-sensitive vs. resistant patient samples. Conclusions: Our data show higher concentration of exosomes and exosomal proteins in OC cases vs controls. Differentially expressed proteins have also been found between samples from platinum-sensitive and platinum-resistant patients. These results suggest that exosomes may be involved in the progression and spread of OC and may provide insights into current treatment response.

The Future of Minimal-Access Myoma Surgery with In-Bag Contained Morcellation.

Contained electromechanical morcellation has emerged as a safety approach for laparoscopic myomatous tissue retrieval. This retrospective single-center analysis evaluated the bag deployment practicability and safety of electromechanical in-bag morcellation when used for big surgical benign specimens. The main age of patients was 39.3 years (range 21 to 71); 804 myomectomies, 242 supracervical hysterectomies, 73 total hysterectomies, and 1 retroperitoneal tumor extirpation were performed. A total of 78.7% of specimens weighed more than 250 g (n = 881) and 9% more than 1000 g. The largest specimens, weighing 2933 g, 3183 g, and 4780 g, required two bags for complete morcellation. Neither difficulties nor complications related to bag manipulation were recorded. Small bag puncture was detected in two cases, but peritoneal washing cytology was free of debris. One retroperitoneal angioleiomyomatosis and three malignancies were detected in histology (leiomyosarcoma = 2; sarcoma = 1); therefore, patients underwent radical surgery. All patients were disease-free at 3 years follow-up, but one patient presented multiple abdominal metastases of the leiomyosarcoma in the third year; she refused subsequent surgery and was lost from follow-up. This large series demonstrates that laparoscopic bag morcellation is a safe and comfortable method to remove large and giant uterine tumors. Bag manipulation takes only a few minutes, and perforations rarely occur and are easy to detect intraoperatively. This technique did not result in the spread of debris during myoma surgery, potentially avoiding the additional risk of parasitic fibroma or peritoneal sarcoma.

Do Exophytic and Endophytic Patterns in Borderline Ovarian Tumors Have Different Prognostic Implications? A Large Multicentric Experience.

Borderline ovarian tumor (BOT) accounts for 15-20% of all epithelial ovarian tumors. Concerns have arisen about the clinical and prognostic implications of BOT with exophytic growth patterns. We retrospectively reviewed all cases of BOT patients surgically treated from 2015 to 2020. Patients were divided into an endophytic pattern (with intracystic tumor growth and intact ovarian capsule) and an exophytic pattern (with tumor growth outside the ovarian capsule) group. Among the 254 patients recruited, 229 met the inclusion criteria, and of these, 169 (73.8%) belonged to the endophytic group. The endophytic group showed more commonly an early FIGO stage than the exophytic group (100.0% vs. 66.7%, p < 0.001). Furthermore, tumor cells in peritoneal washing (20.0% vs. 0.6%, p < 0.001), elevated Ca125 levels (51.7% vs. 31.4%, p = 0.003), peritoneal implants (0 vs. 18.3%, p < 0.001), and invasive peritoneal implants (0 vs. 5%, p = 0.003) were more frequently observed in the exophytic group. The survival analysis showed 15 (6.6%) total recurrences, 9 (5.3%) in the endophytic and 6 (10.0%) patients in the exophytic group (p = 0.213). At multivariable analysis, age (p = 0.001), FIGO stage (p = 0.002), fertility-sparing surgery (p = 0.001), invasive implants (p = 0.042), and tumor spillage (p = 0.031) appeared significantly associated with recurrence. Endophytic and exophytic patterns in borderline ovarian tumors show superimposable recurrence rates and disease-free survival.

The role of cytology in patients undergoing pressurized intraperitoneal aerosol chemotherapy (PIPAC) treatment for peritoneal carcinomatosis.

Cytology of ascites or peritoneal washing is a routine part of staging of peritoneal metastases (PM). We aim to determine value of cytology in patients undergoing pressurized intraperitoneal aerosol chemotherapy (PIPAC). Single-center retrospective cohort study included consecutive patients having PIPAC for PM of different primary between January 2015 and January 2020. A total of 75 patients (median 63 years (IQR 51-70), 67 % female) underwent a total of 144 PIPAC. At PIPAC 1 59 % patients had positive and 41 % patients had negative cytology. Patients with negative and positive cytology only differed in terms of symptoms of ascites (16% vs. 39 % respectively, p=0.04), median ascites volume (100 vs. 0 mL, p=0.01) and median PCI (9 vs. 19, p<0.01). Among 20 patients who completed 3 PIPACs (per protocol), cytology changed in one from positive to negative, and in two from negative to positive. Median overall survival was 30.9months in the per protocol group and 12.9months in patients having <3 PIPACs (=0.519). Positive cytology under PIPAC treatment is more frequently encountered in patients with higher PCI and symptomatic ascites. Cytoversion was rarely observed and cytology status had no impact on treatment decisions in this cohort.

MALIGNANT STRUMA OVARII (KASUS LANGKA)

Struma ovarii (SO) are rare and accur in 2-3% of mature ovarian teratomas, most SO cases are benign. Malignant struma ovarii (MSO) is even more rare and occurs in 0,5- 5% of all cases, with the most common histologies identified are papillary and follicular carcinoma, most commonly affect patients in their 4th to 6th decade of life. Due to the rarity of malignant transformation, data about its management are lacking. A 49-years-old woman P2A0 with abdominal pain and pelvic mass, admitted for a hysterectomy and bilateral salpingo-oophorectomy. The left ovary was measured 13x11x5 cm, cystic and partly solid, filled with gelatinous fluid, composed of thyroid follicles with the appearance of "ground glass", "nuclear inclusion" and "nuclear groove", forming a papillifer structure. The diagnostic of MSO is based on histological features include cellular atypia, increased hyperplasia, nuclear pleomorphism, mitotic activity, vascular and/or capsular invasion. To be classified as a MSO, teratoma containing 50% or more thyroid tissue. MSO necessitates surgical staging for ovarian cancer with pelvic washings, total abdominal hysterectomy, bilateral salpingo- oophorectomy, lymph node sampling, total thyroidectomy, and radioactive I-131 ablation. Based on the clinical examination and histopatological examination the patient was diagnosed with MSO (ICD O: 9090/3).

Survival outcomes and the prognostic significance of clinicopathological features in patients with endometrial clear cell carcinoma: a 35-year single-center retrospective study

BackgroundTo evaluate the oncological outcomes and the impact of clinicopathological factors on endometrial clear cell carcinoma (ECCC) outcomes.MethodsMedical records of patients with primary ECCC treated at our center between 1985 and December 2020 were reviewed. Overall survival (OS) and progression-free survival (PFS) were the endpoints. The Kaplan–Meier method and Cox regression analysis were used.ResultsIn total, 156 patients were included, of whom 59% and 41% had early- and advanced-stage ECCC, respectively. The median age of onset was 61 years, and 80.8% of the patients were postmenopausal. Ninety-two (59%) and 64 (41%) patients had pure ECCC and mixed endometrial carcinoma with clear cell carcinoma (CCC) components, respectively. Mixed pathological components, elevated cancer antigen 125 levels, positive lymphovascular space invasion, deep myometrial invasion, and malignant peritoneal washing cytology (PWC) were more frequently observed in the advanced stage. Thirty-nine patients (25%) experienced relapse and 32 patients (20.5%) died. The 5-year PFS and OS rates for the entire cohort were 72.6% and 79%, respectively. Multivariate analysis showed that advanced-stage disease and positive PWC significantly decreased PFS, while advanced-stage disease and older age (> 61 years) significantly decreased OS.ConclusionsECCC is a rare and aggressive type II endometrial carcinoma that is common in older women and patients with advanced-stage disease. Positive PWC was associated with decreased PFS, although its presence did not influence the stage. Positive PWC, and advanced stage and older age were independent negative prognostic factors.

Clinicopathological Parameters Associated With Peritoneal Involvement in Epithelial Ovarian Tumors.

Introduction Ovarian tumors remain one of the leading malignancies of the female genital tract, with a high mortality rate due to their insidious onset and lack of detection at an earlier stage. These tumors metastasize by direct extension into the neighboring pelvic organs; hence, the detection of peritoneal metastasis is valuable for staging and prognostic purposes. Peritoneal wash cytological analysis is an effective predictor of the involvement of the ovarian surface and peritoneal dissemination even in subclinical involvement of the peritoneum. The study aims to determine the significance of peritoneal wash cytology as a prognostic parameter and correlate it with various clinicohistological features. Methods A retrospective study was conducted at the Department of Histopathology, Liaquat National Hospital, Karachi, Pakistan, between July 2017 and June 2022. During this period, all the cases of ovarian tumors (borderline and malignant) that underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy and omental and lymph node sampling were included in the study. After opening the abdominal cavity, the free fluid present was aspirated immediately, the peritoneum was lavaged with 50-100 mL of warm saline, and samples were collected and sent for cytological analysis. Four cytospin smear slides and cell block preparation were prepared. The findings of peritoneal cytology were correlated with various clinicohistological features. Results A total of 118 cases of ovarian tumors were included in the study. Serous carcinoma was the most common sub-type (50.8%), followed by endometrioid carcinoma (14.4%), and the mean age at diagnosis was found to be 49.9±14.9 years. The mean tumor size was 11.2 cm. The majority of the cases of ovarian carcinoma were of high grade (78.8%), with capsular invasion present in 61% of cases. Positive peritoneal cytology was noted in 58.5% of cases, with omental involvement in 52.5% of cases. Serous carcinoma showed the highest frequency of positive cytology (69.6%) and omental metastasis (74.2%). Apart from tumor type, positive peritoneal cytology showed a significantly positive correlation with age, tumor grade, and capsular invasion. Conclusion Based on our study findings, we conclude that peritoneal wash cytology is a sensitive indicator of the peritoneal spread of ovarian carcinoma, with a significant prognostic value. Serous carcinomas, especially high-grade with capsular invasion, were found to be predictors of peritoneal involvement of ovarian tumors. Although we found smaller tumors to be associated with peritoneal disease more compared to larger ones, this most likely is attributed to tumor histology, as larger tumors were most commonly mucinous compared to serous carcinomas.

Cytological features of mesonephric-like adenocarcinoma of the uterine corpus.

Although several studies have documented the histological features of uterine mesonephric-like adenocarcinoma (MLA), its cytological features have been rarely reported. We searched for histologically confirmed uterine MLA cases in the pathology archives of three institutions between 2010 and 2021. All available cytology slides were examined to identify the cytological features of uterine MLA. We included 16 patients with uterine MLA and reviewed the slides obtained from 21 cytology samples. Samples were obtained from the cervicovagina (9/21, 42.9%), peritoneal washing (8/21, 38.1%), pleural effusion (2/21, 9.5%), and transbronchial needle aspiration of mediastinal lymph node (2/21, 9.5%). Preparation methods included ThinPrep (11/21, 52.4%), SurePath (8/21, 38.1%), and conventional smear (2/21, 9.5%). Regardless of the sampling site and preparation method, cytology samples displayed tight three-dimensional cellular clusters showing monotonous, small-to-medium-sized, round, hyperchromatic nuclei, indistinct nucleoli, scant cytoplasm, and high nuclear-to-cytoplasmic ratio. Approximately half of the samples (10/21, 47.6%) showed hyaline-like globules. Mitotic figures (7/21, 33.3%) and apoptotic bodies (13/21, 61.9%) were also observed. No tumor diathesis or nuclear feathering was identified. Irrespective of sampling site and preparation method, the majority of uterine MLA cases showed the following cytological features: tight three-dimensional cellular clusters showing small-to-medium-sized, round, hyperchromatic nuclei with indistinct nucleoli and high nuclear-to-cytoplasm ratio. In case a cytology sample suspicious of a glandular lesion displays these cytological features, which are distinct from those of endocervical adenocarcinoma, uterine MLA should be included in the differential diagnosis.

A case of diffusely infiltrating colorectal cancer with distinctive endoscopic and histologic findings

A man in his 50s visited our hospital for positive fecal immunochemical tests and transient abdominal pain. Colonoscopy revealed multiple diverticula and localized reddish, granular mucosa with mildly converging folds at the ascending colon (Fig. 1). Narrow-band imaging magnifying endoscopy visualized normal pits (Kudo’s type I pit pattern) on the reddish mucosa (Fig. 2). Endoscopically, the findings were not suggestive of neoplastic changes. Malignancy was not proven by biopsy samples.Figure 2NBI magnifying endoscopy visualized normal pits on the reddish mucosa.View Large Image Figure ViewerDownload Hi-res image Download (PPT) One year later, he underwent colonoscopy again after another positive fecal immunochemical test. The ascending colon became severely strictured because of worsening converging folds. An enlargement was seen in the reddish mucosa, but ulcerations were absent (Fig. 3). Adenocarcinoma cells were detected in the biopsy specimens. Other malignant neoplasms were not revealed in the chest and abdomen by EGD and contrast-enhanced CT; thus, the lesion was considered to be primary type 4 (the Paris endoscopic classification) ascending colon cancer. The patient was transferred to a tertiary referral institution. Although the preoperative TNM stage was cT3N0M1c cStage IVc, intraoperative peritoneal washing cytology was negative. Therefore, a right hemicolectomy was performed. The resected tumor was 31 × 20 mm in size. Histologically, the diffuse spread of well to moderately differentiated adenocarcinoma cells were observed, accompanied by significant desmoplastic reactions (Figs. 4 and 5). Although the tumor partially infiltrated into the subserosal layer, most of the mucosal surface was covered with normal glands, having only focal cancer exposure (Fig. 6). The patient was finally diagnosed with diffusely infiltrating ascending colon cancer with predominantly differentiated histology.Figure 5The higher-power field image of arrow on Figure 4 (H&E, orig. mag. ×30).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Most of the mucosal surface was covered with normal glands. Carcinoma cells were only focally exposed (arrowhead on Figure 4; H&E, orig. mag. ×50).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The initial endoscopic findings seemed to reflect those of diffusely infiltrating colorectal cancer at a relatively early stage and should be noted. The chronological changes mentioned above also shed some light on the growth pattern of this particular disease. This case was considered to be distinctive from the perspective of endoscopic and histologic features. All authors disclosed no financial relationships.

All-Trans Retinoic Acid Promotes M2 Macrophage Polarization in Vitro by Activating the p38MAPK/STAT6 Signaling Pathway

ABSTRACT Background M2-type macrophages are inflammation-suppressing cells that are differentiated after induction by cytokines such as IL-4 or IL-13, which play an important regulatory role in inflammation and influence the regression of inflammation-related diseases. All-trans retinoic acid (ATRA) has an important role in suppressing immune-mediated inflammatory responses but the effect and underlying mechanism of ATRA on the polarization of M2 macrophages remains unclear. Methods Macrophages were isolated from peritoneal wash fluid, and IL-4 (20 ng/mL) was used to construct a m2-type macrophage polarization model. The model was incubated with different concentrations of ATRA (15 µg/ml, 30 µg/ml, 45 µg/ml) for 24 h, and pretreated macrophages with p38MAPKα inhibitor SB202190 (20 μM). MTT, Trypan blue staining, Annexin V-PE/7-AAD staining, flow cytometry, real-time PCR and western blotting were used to investigate the effect and mechanism of ATRA on the polarization of M2 macrophages. Results Compared with the IL-4 group, the proportion of F4/80+CD206+ M2-type macrophages was significantly higher in the ATRA group (P < 0.01). mRNA and protein expression levels of Arg-1, IL-10 and TGF-β1 were as significantly higher (P < 0.01) in the ATRA group as phosphorylation levels of STAT6 and p38MAPK (P < 0.01). After pretreatment with the addition of the inhibitor SB202190, M2-type macrophages proportion and their associated factors expression were significantly (P < 0.01) reduced, as compared with those in the ATRA group, but they were comparable (P > 0.05) with the IL-4 group. Conclusion The combination of ATRA and IL-4 activated the p38MAPK/STAT6-signaling pathway to promote polarization of M2 macrophages.

Efficacy of a Novel Pleiotropic MMP-Inhibitor, CMC2.24, in a Long-Term Diabetes Rat Model with Severe Hyperglycemia-Induced Oral Bone Loss.

CMC2.24, a novel 4-(phenylaminocarbonyl)-chemically-modified-curcumin, is a pleiotropic MMP-Inhibitor of various inflammatory/collagenolytic diseases including periodontitis. This compound has demonstrated efficacy in host modulation therapy along with improved resolution of inflammation in various study models. The objective of current study is to determine the efficacy of CMC2.24 in reducing the severity of diabetes, and its long-term role as an MMP-inhibitor, in a rat model. Twenty-one adult male Sprague-Dawley rats were randomly distributed into three groups: Normal (N), Diabetic (D) and Diabetic+CMC2.24 (D+2.24). All three groups were orally administered vehicle: carboxymethylcellulose alone (N, D), or CMC2.24 (D+2.24; 30mg/kg/day). Blood was collected at 2-months and 4-months' time-point. At completion, gingival tissue and peritoneal washes were collected/analyzed, and jaws examined for alveolar bone loss by micro-CT. Additionally, sodium hypochlorite(NaClO)-activation of human-recombinant (rh) MMP-9 and its inhibition by treatment with 10μM CMC2.24, Doxycycline, and Curcumin were evaluated. CMC2.24 significantly reduced the levels of lower-molecular-weight active-MMP-9 in plasma. Similar trend of reduced active-MMP-9 was also observed in cell-free peritoneal and pooled gingival extracts. Thus, treatment substantially decreased conversion of pro- to actively destructive proteinase. Normalization of the pro-inflammatory cytokine (IL-1ß, resolvin-RvD1), and diabetes-induced osteoporosis was observed in presence of CMCM2.24. CMC2.24 also exhibited significant anti-oxidant activity by inhibiting the activation of MMP-9 to a lower-molecular-weight (82kDa) pathologically active form. All these systemic and local effects were observed in the absence of reduction in severity of hyperglycemia. CMC2.24 reduced activation of pathologic active-MMP-9, normalized diabetic osteoporosis, and promoted resolution of inflammation but had no effect on the hyperglycemia in diabetic rats. This study also highlights the role of MMP-9 as an early/sensitive biomarker in the absence of change in any other biochemical parameter. CMC2.24 also inhibited significant activation of pro-MMP-9 by NaOCl (oxidant) adding to known mechanisms by which this compound treats collagenolytic/inflammatory diseases including periodontitis.

Ex vivo oncolytic and immune activity of CF33-hNIS-antiPDL1 against fresh peritoneal cells from patients with gastric cancer with and without peritoneal metastases.

430 Background: No effective therapy exists for peritoneal metastases (PM) from gastric cancer (GC), which remain fatal within months of diagnosis. We investigated a novel chimeric oncolytic virus platform, CF33-OV, for its anti-cancer potential against peritoneal cancer cells from GC patients. Methods: We prospectively collected fresh ascites or peritoneal washings from 30 GC patients between 2019 and 2020. These included 16 males (53.3%) and 14 females (46.7%) with a mean age of 51.3 ± 15.1 years. Of the 30 patients, 23 (90%) had cytology or biopsy-proven PM. Cells isolated from the peritoneal fluid were analyzed using flow cytometry for immune cell markers and/or treated with PBS, CF33-GFP (MOI=3), or CF33-hNIS-antiPDL1 (MOI=3) for 15 hours. We evaluated the treated cells for virus infection, replication, CD8+ and CD4+ expression with exhaustion markers, virus infection, replication, and cytotoxicity. We evaluated ex vivo CD274 (PD-L1) blockade by virus-encoded anti-PD-L1 scFv and the release of growth factors and cytokines following CF33-hNIS-antiPDL1 treatment. Results: The CD45- cancer cell percentage was higher, and the CD8+ T cell percentage was significantly lower in the ascites group than in the peritoneal washing group. At baseline, low levels of CD274 (PD-L1), CD252 (OX40L), and EGFR were identified on the surface of cancer cells. CD4+ and CD8+ T cells did not express cell surface exhaustion markers. After 15 hours of treatment, CF33-GFP infected, replicated in, and killed cancer cells ex vivo with a significantly higher dead cell percentage than the PBS control group (10.8% treated vs. 3.1% control, p-value &lt;0.05). Of the live cancer cells, 69% were GFP+ by flow cytometry. Fluorescence microscopy results confirmed virus-encoded GFP expression in the ex vivo cells. Moreover, CF33-hNIS-antiPDL1 produced functional anti-PD-L1 scFv, which blocked phycoerythrin labeled anti-PD-L1 binding to PD-L1 on live cancer cells. Virus treatment increased CD8+CD107α+ and CD4+CD107α+ T cell percentages while decreasing the release of growth factors (EGF and PDGF) and inhibitory immune factors (soluble anti-PD-L1, GM-CSF, and IL-10). Conclusions: CF33-hNIS-antiPDL1 is oncolytic with immune-activating effects against GCPM ex vivo and has solid potential for intraperitoneal therapy of GCPM patients.