Atlas of Tumor and Tumor Microenvironment Cells of Lymphovascular Space Invasion (LVSI) in High-Grade Serous Endometrial Adenocarcinoma: A Case Study.

Lymphovascular invasion (LVSI) is defined as the presence of tumor cells within a definite endothelial-lined space (lymphatics or blood vessels) in the organ surrounding invasive carcinoma. The presence of LVI is associated with an increased risk of lymph nodes and distant metastases. Lymphovascular invasion is described as cancer within blood or lymph vessels and is an independent risk factor for metastasis, recurrence, and mortality. This study aims to present the marker-based immunohistological characterization of cells around LVSI in a high-grade adenocarcinoma of the endometrium to build a cellular atlas of cells of LVSI. A cellular characterization of the cells around lymphovascular space invasion in a 67-year-old female patient with invasive high-grade serous endometrial adenocarcinomas is presented. Resected tumor tissue from a consented patient with invasive high-grade serous endometrial adenocarcinoma was obtained within an hour of surgery. The expressions of the epithelial markers (CK8, 18, and EpCAM), LCA (leukocyte common antigen) marker (CD45), proliferation marker (Ki67), apoptosis markers (cleaved PARP and cleaved caspase3), immune cell markers (CD3, CD4, CD8, CD56, CD68, CD163, FoxP3, PD-1, PD-L1), pro-inflammatory marker (IL-12-RB2), and fibroblast/mesenchyme markers (S100A7, SMA, and TE-7) of the resected tissue on the IHC stains were evaluated and scored by a pathologist. Acknowledging the deterministic role of LVSI in a high-grade adenocarcinoma of the endometrium, our study presents the first marker-based immunohistological atlas of the tumor and TME compartments in the context of epithelial cell markers, proliferation markers, apoptosis markers, macrophage markers, and fibroblast markers. Our study demonstrates that an aggressive disease like a high-grade adenocarcinoma of the endometrium inflicts the pro-metastatic event of LVSI by involving the immune landscape of both tumor and TME. This study demonstrates, for the first time, that the tumor cells within LVSI are positive for IL-12R-B2 and S100A4.

Tumor-TME Bipartite Landscape of PD-1/PD-L1 in Endometrial Cancers.

The bipartite landscape of tumor cells and stromal cells determines a tumor's response to treatment during disease management. In endometrial cancers (ECs), the mechanistic contribution of PD-L1/L2 and PD-1 signaling of the host's tumor microenvironment (TME) (CAF and immune cells) in the context of the tumor cells is elusive. To understand the tumor-stroma-immune crosstalk, we studied the compartmental pattern of PD-L1/L2 and PD-1 expression in EC tissues and their matched CAFs. Over 116 surgically resected tumors (T) and the tumor-adjacent normal tissues (N) were obtained from consented unselected consecutive patients. IHC was performed in T, N-epi-thelium, and the stromal mesenchymal environment (SME; mesenchyme) in the T and N tissues. The staining intensity and distribution patterns of PD-L1/L2 and PD-1 in the FFPE sections of T and N were evaluated by a pathologist using a standard scoring system of TPS and CPS. We tested the PD-L1/L2 and PD-1 immune landscape of tumor-TME pair and normal epithelial-stromal mesenchyme pairs from patients with different grades of disease vis-à-vis their CAF PD-L1 levels. We used qRT-PCR to determine the expressions of mRNAs, while the flow cytometry and ICC determined the level of expression of proteins. We observed higher levels of PD-L1 mRNA and protein expression in primary CAFs from the resected tumor tissue compared to the tumor-adjacent normal tissues. We also determined the expression of patients' soluble PD-L1/L2 as peripheral readouts of PD-L1/L2 and PD-1. As we evaluated the results in the context of their pathological parameters, such as grades, stages, lymphovascular invasion, percentage of myometrial invasion, and dMMR in patients, the dominance of PD-L1 expression in TME was positively correlated to the higher pathological grades of tumors, and its relationship with the dMMR. Since the neutralization of CD8-positive cytotoxic T-cells is PD-L1-dependent, our data indicate that irrespective of the PD-L1 positivity of tumor cells, the PD-L1-positive CAFs can play a critical role in bringing out an additional load of PD-L1 for an effective engagement of PD-1 within a tumor mass.

A CAF-Based Two-Cell Hybrid Co-Culture Model to Test Drug Resistance in Endometrial Cancers.

The management of advanced or recurrent endometrial cancers presents a challenge due to the development of resistance to treatments. The knowledge regarding the role of the tumor microenvironment (TME) in determining the disease's progression and treatment outcome has evolved in recent years. As a TME component, cancer-associated fibroblasts (CAFs) are essential in developing drug-induced resistance in various solid tumors, including endometrial cancers. Hence, an unmet need exists to test the role of endometrial CAF in overcoming the roadblock of resistance in endometrial cancers. We present a novel tumor-TME two-cell ex vivo model to test CAF's role in resisting the anti-tumor drug, paclitaxel. Endometrial CAFs, both NCAFs (tumor-adjacent normal-tissue-derived CAFs) and TCAFs (tumor-tissue-derived CAFs) were validated by their expression markers. Both TCAFs and NCAFs expressed positive markers of CAF, including SMA, FAP, and S100A4, in varying degrees depending on the patients, while they consistently lacked the negative marker of CAF, EpCAM, as tested via flow cytometry and ICC. CAFs expressed TE-7 and immune marker, PD-L1, via ICC. CAFs better resisted the growth inhibitory effect of paclitaxel on endometrial tumor cells in 2D and 3D formats compared to the resistance of the tumoricidal effect of paclitaxel in the absence of CAFs. TCAF resisted the growth inhibitory effect of paclitaxel on endometrial AN3CA and RL-95-2 cells in an HyCC 3D format. Since NCAF similarly resisted the growth inhibitor action of paclitaxel, we tested NCAF and TCAF from the same patient to demonstrate the protective action of NCAF and TCAF in resisting the tumoricidal effect of paclitaxel in AN3CA in both 2D and 3D matrigel formats. Using this hybrid co-culture CAF and tumor cells, we established a patient-specific, laboratory-friendly, cost-effective, and time-sensitive model system to test drug resistance. The model will help test the role of CAFs in developing drug resistance and contribute to understanding tumor cell-CAF dialogue in gynecological cancers and beyond.

Open Latarjet with Metal-Free Cerclage Fixation.

Despite multiple modifications, the Latarjet is still the most popular procedure for recurrent anterior shoulder instability with glenoid bone loss. Partial or subtotal resorption of the graft is common, potentially leading to hardware prominence and risk of anterior soft-tissue impingement. To minimize the technical difficulties and morbidity associated with metallic implants, a coracoid and conjoint tendon transfer with a mini-open approach using Cerclage tape suture is described, as an alternative for the Latarjet procedure typically performed with metal screws and plates.

Characterization and Clinical Relevance of Endometrial CAFs: Correlation between Post-Surgery Event and Resistance to Drugs.

Cancer-associated fibroblasts (CAFs) within a solid tumor can support the progression of cancer. We studied the identification and characterization of patient-derived endometrial CAFs in the context of their clinical relevance in endometrial cancers. We established patient-derived primary cultures of CAFs from surgically resected tumors (TCAF) and tumor-adjacent normal (NCAF) tissues in 53 consented patients with success rates of 97.7% and 75%, respectively. A passage of CAF was qualified by the (1) absence of CK 8,18,19, EpCAM, CD45, and CD31, and (2) presence of SMAalpha, S100A4, CD90, FAP, TE-7, CD155, PD-L1, TGFB, PDGFRA (qRT-PCR, flow cytometry, Western blot, ICC). Out of the 44 established CAFs, 31 were aggressive (having an early, i.e., 4-7 week, establishment time and/or >3 passages) compared to 13 which were non-aggressive. A post-surgery-event (PSE) was observed in 7 out of 31 patients bearing aggressive CAFs, 2 of whom were also positive for CTCs, while none of the 13 patients bearing non-aggressive CAFs had events. A positive correlation was found between patients with grade 3 (p = 0.025) as well as stage 3/4 diseases (p = 0.0106) bearing aggressive CAFs and the PSE. Finally, aggressive TCAFs from patients with PSE resisted the effects of paclitaxel and lenvatinib on the growth of HUVEC and endometrial tumor cells. Our study is the first to report a correlation between the PSE and the aggressive nature of CAFs in endometrial cancers and provides an undeniable reason to study the in-depth mechanism of CAF function towards the development of treatment resistance in endometrial cancers.

Patient-Derived Primary Cancer-Associated Fibroblasts Mediate Resistance to Anti-Angiogenic Drug in Ovarian Cancers.

Ovarian cancers rank first in both aggressiveness and dismal prognosis among gynecological neoplasms. The poor outcome is explained by the fact that most patients present with late-stage disease and progress through the first line of treatment. Ovarian neoplasms, especially epithelial ovarian cancers, are diagnosed at advanced/metastatic stages, often with a high angiogenesis index, one of the hallmarks of ovarian cancers with rapid progression and poor outcome as resistance to anti-angiogenic therapy develops. Despite therapy, the metastatic progression of aggressive ovarian cancer is a spectacularly selective function of tumor cells aided and abetted by the immune, mesenchymal and angiogenic components of the tumor microenvironment (TME) that enforces several pro-metastatic event(s) via direct and indirect interactions with stromal immune cells, cancer-associated fibroblasts (CAFs), and vascular endothelial cells. Since transdifferentiation of tumor endothelium is one of the major sources of CAFs, we hypothesized that ovarian CAF plays a critical role in resisting anti-angiogenic effects via direct crosstalk with endothelium and hence plays a direct role in the development of resistance to anti-angiogenic drugs. To test the hypothesis, we set up a hybrid ex vivo model for co-culture comprising Patient-Derived ex vivo primary CAFs from ovarian tumor samples and human umbilical vein endothelial cells (HUVEC). Patient-Derived CAFs were characterized by the mRNA and protein expression of positive (SMA, S100A4, TE-7, FAP-A, CD90/THY1), negative (EpCAM, CK 8,18, CD31, CD44, CD45), functional (PDGFRA, TGFB1, TGFB2, TGFRA) and immunological markers (PD-L1, PD-L2, PD-1) associated with CAFs by qRT-PCR, flow cytometry, Western blot, and ICC. Data from our HUVEC-on-CAF ex vivo Hybrid Co-Culture (HyCC) study demonstrate the pro-angiogenic effect of Patient-Derived ovarian CAFs by virtue of their ability to resist the effect of anti-angiogenic drugs, thereby aiding the development of resistance to anti-angiogenic drugs. Ascertaining direct experimental proof of the role of CAFs in developing resistance to specific anti-angiogenic drugs will provide an opportunity to investigate new drugs for counteracting CAF resistance and "normalizing/re-educating" TME in aggressive ovarian cancers. Our data provide a unique experimental tool for the personalized testing of anti-angiogenic drugs, positively predicting the development of future resistance to anti-angiogenic drugs well before it is clinically encountered in patients.

Identification and Morphological Characterization of Features of Circulating Cancer-Associated Macrophage-like Cells (CAMLs) in Endometrial Cancers.

The blood of patients with solid tumors contains circulating tumor-associated cells, including epithelial cells originating from the tumor mass, such as circulating tumor cells (CTCs), or phagocytic myeloid cells (differentiated monocytes), such as circulating cancer-associated macrophage-like cells (CAMLs). We report for the first time the identification and in-depth morphologic characterization of CAMLs in patients with endometrial cancers. We isolated CAMLs by size-based filtration on lithographically fabricated membranes followed by immunofluorescence, using a CD45+/CK 8,18,19+/EpCAM+/CD31+/macrophage-like nuclear morphology, from > 70 patients. Irrespective of the histological and pathological parameters, 98% of patients were positive for CAMLs. Two size-based subtypes of CAMLs, <20 µm (tiny) and >20 µm (giant) CAMLs, of distinctive polymorphic morphologies with mononuclear or fused polynuclear structures in several morphological states were observed, including apoptotic CAMLs, CAML−WBC doublets, conjoined CAMLs, CAML−WBC clusters, and CTC−CAML−WBC clusters. In contrast, CAMLs were absent in patients with non-neoplastic/benign tumors, healthy donors, and leucopaks. Enumerating CTCs simultaneously from the same patient, we observed that CTC-positive patients are positive for CAMLs, while 55% out of all CAML-positive patients were found positive for CTCs. Our study demonstrated for the first time the distinctive morphological characteristics of endometrial CAMLs in the context of the presence of CTCs in patients.

A Laboratory-Friendly CTC Identification: Comparable Double-Immunocytochemistry with Triple-Immunofluorescence.

The source of circulating tumor cells (CTC) in the peripheral blood of patients with solid tumors are from primary cancer, metastatic sites, and a disseminated tumor cell pool. As 90% of cancer-related deaths are caused by metastatic progression and/or resistance-associated treatment failure, the above fact justifies the undeniable predictive and prognostic value of identifying CTC in the bloodstream at stages of the disease progression and resistance to treatment. Yet enumeration of CTC remains far from a standard routine procedure either for post-surgery follow-ups or ongoing adjuvant therapy. The most compelling explanation for this paradox is the absence of a convenient, laboratory-friendly, and cost-effective method to determine CTC. We presented a specific and sensitive laboratory-friendly parallel double-detection format method for the simultaneous isolation and identification of CTC from peripheral blood of 91 consented and enrolled patients with various malignant solid tumors of the lung, endometrium, ovary, esophagus, prostate, and liver. Using a pressure-guided method, we used the size-based isolation to capture CTC on a commercially available microfilter. CTC identification was carried out by two expression marker-based independent staining methods, double-immunocytochemistry parallel to standard triple-immunofluorescence. The choice of markers included specific markers for epithelial cells, EpCAM and CK8,18,19, and exclusion markers for WBC, CD45. We tested the method's specificity based on the validation of the staining method, which included positive and negative spiked samples, blood from the healthy age-matched donor, healthy age-matched leucopaks, and blood from metastatic patients. Our user-friendly cost-effective CTC detection technique may facilitate the regular use of CTC detection even in community-based cancer centers for prognosis, before and after surgery.

Convalescent Plasma in COVID-19. Mortality-Safety First Results of the Prospective Multicenter FALP 001-2020 Trial

BackgroundThe use of convalescent plasma (CP) to treat COVID-19 has shown promising results; however, its effectiveness remains uncertain. The purpose of this study was to determine the safety and mortality of CP among patients hospitalized with COVID-19. Study Design and MethodsThis multicenter, open-label, uncontrolled clinical trial is currently being conducted at nine hospitals in Chile. Patients hospitalized due to COVID-19 who were still within 14 days since symptom onset were classified into four groups: Patients with cancer and severe COVID-19. Patients with cancer and non-severe COVID-19. Patients with severe COVID-19 and patients with non-severe COVID-19 only. The intervention involved two 200-cc. CP transfusions with anti-SARS-CoV-2 IgG titers [≥] 1:320 collected from COVID-19-recovered donors. Results192 patients hospitalized for COVID-19 received CP transfusions. At the first transfusion, 90.6% fulfilled the criteria for severity, and 41.1% required mechanical ventilation. 11.5% of the patients had cancer. Overall 7-day and 30-day mortality since the first CP transfusion was 5.7% and 16.1% respectively. There were no differences at either time point in mortality between the four groups. Patients on mechanical ventilation when receiving CP had higher mortality rates than those who were not (22.8% vs. 11.5%; p = 0.037). Overall 30-day mortality was higher in patients over 65 than in younger patients (p = 0.019). Severe adverse events were reported in four patients (2.1%) with an overall transfusion-related lung injury rate of 1.56%. No CP-related deaths occurred. DiscussionCP is safe when used in patients with COVID-19 even when also presenting severity criteria or risk factors. Our mortality rate is comparable to reports from larger studies. Controlled clinical trials are required to determine efficacy. RegistrationNCT04384588

Viral shedding dynamics reveals sputum as a reliable and cost-saving specimen for SARS-CoV-2 diagnosis within the first 10 days since symptom onset: A prospective cohort study

BackgroundCoronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome virus (SARS-CoV-2) is challenging global public health, due to an increasing demand for testing and the shortage of diagnostic supplies. Nasopharyngeal swab (NPS) is considered the optimal sample for SARS-CoV2 diagnosis and sputum (SPT) has been proposed as an economic alternative. However, the temporal concordance of diagnosis in NPS and SPT has not been addressed. MethodsThrough a longitudinal study we compared the shedding dynamics of SARS-CoV-2 RNA evaluated by RT-qPCR in serially collected SPT and NPS obtained from 82 ambulatory and hospitalized patients during acute infection and convalescence. The concordance during the follow-up and cost analysis between both collected specimens was evaluated. FindingsWe analyzed 379 samples, 177 NPS and 202 SPT. The highest proportion of positive samples was detected within the first 15 days after the symptoms onset. The median time of positivity was higher for NPS (median= 25 days) than SPT (median= 21 days). There was no significant difference in the median RT-qPCR CT values between both sample types. The temporal categorization of matched-paired samples indicated substantial correlation (r=0{middle dot}6023) and substantial agreement (87{middle dot}23%) during the first ten days since symptoms onset (kappa = 0{middle dot}697). A cost analysis demonstrated a significant saving when the SPT specimen was used. InterpretationSputum is a feasible and cost-saving alternative to NPS, providing an equivalent value for the detection and follow-up of SARS-CoV-2 RNA. FundingAgencia Nacional de Investigacion y Desarrollo (ANID) of Chile, NIH-NIAID USA.

Early Anti-SARS-CoV-2 Convalescent Plasma in Patients Admitted for COVID-19: A Randomized Phase II Clinical Trial

Background: Convalescent plasma (CP), despite limited evidence on its efficacy, is being widely used as a compassionate therapy for hospitalized patients with COVID-19. We aimed to evaluate the efficacy and safety of early CP therapy in COVID-19 progression. Methods: Open-label, single-center, randomized clinical trial performed in an academic center in Santiago, Chile from May 10, 2020, to July 18, 2020, with final follow-up August 17, 2020. The trial included patients hospitalized within the first 7 days of COVID-19 symptoms onset, presenting risk factors for illness progression and not on mechanical ventilation. The intervention consisted in immediate CP (early plasma group) versus no CP unless developing pre-specified criteria of deterioration (deferred plasma group). Additional standard treatment was allowed in both arms. The primary outcome was a composite of mechanical ventilation, hospitalization for >14 days or death. Key secondary outcomes included: time to respiratory failure, days of mechanical ventilation, hospital length-of-stay, mortality at 30 days, and SARS-CoV-2 RT-PCR clearance rate. Results: Of 58 randomized patients (mean age, 65.8 years, 50% male), 57 (98.3%) completed the trial. A total of 13 (43.3%) participants from the deferred group received plasma based on clinical aggravation. We found no benefit in the primary outcome (32.1% vs 33.3%, OR 0.95, 95% CI 0.32-2.84, p>0.99) in the early versus deferred CP group. In-hospital mortality rate was 17.9% vs 6.7% (OR 3.04, 95% CI 0.54-17.2, p=0.25), mechanical ventilation 17.9% vs 6.7% (OR 3.04, 95% CI 0.54-17.2, p=0.25), and prolonged hospitalization 21.4% vs 30% (OR 0.64, 95%CI, 0.19-2.1, p=0.55) in early versus deferred CP group, respectively. Viral clearance rate on day 3 (26% vs 8%, p=0.20) and day 7 (38% vs 19%, p=0.37) did not differ between groups. Two patients experienced serious adverse events within 6 or less hours after plasma transfusion. Conclusion: Immediate addition of CP therapy in early stages of COVID-19 -compared to its use only in case of patient deterioration- did not confer benefits in mortality, length of hospitalization or mechanical ventilation requirement.

Normal Intracranial Translucency Values During the First Trimester of Gestation in a Latin American Population.

OBJECTIVES: The purpose of this study was to establish intracranial translucency reference values in healthy fetuses from a Latin American population. METHODS: This work was a cross-sectional retrospective correlational study. A review of sonographic reports from women between gestational ages of 11 weeks and 13 weeks 6 days at 2 health institutes in Bogota, Colombia, whose fetuses had a crown-rump length of 45 to 84 mm was conducted between January 1, 2010, and December 31, 2012. Women with multiple fetuses or with a deceased fetus were excluded. RESULTS: Data corresponding to 1520 obstetric sonographic examinations were included in the statistical analysis. The crown-rump length was between 45 and 84 mm, with a median of 65 mm (interquartile range, 58-73 mm). The median intracranial translucency was 1.7 mm (interquartile range, 1.4-2.2 mm). Different percentiles (1st, 5th, 10th, 25th, 50th, 75th, 90th, 95th, and 99th) were established for each crown-rump length value. A correlation between crown-rump length and intracranial translucency was found, which seemed to be linear. The intracranial translucency value was not significantly correlated with the frontomaxillary angle but was correlated with nuchal translucency, nasal bone length, and metopic suture length. CONCLUSIONS: We present normal intracranial translucency values in the first trimester of single gestations with live fetuses in a Latin American population. These values are similar to those described in other populations. The intracranial translucency value was linearly correlated with crown-rump length in fetuses between gestational ages of 11 weeks and 13 weeks 6 days, which is consistent with previous publications, although these data cannot be interpreted independently.

Factores Psicológicos que Influyen en la Incorporación a la Universidad del Adulto Mayor del Municipio Bauta / Psychological Factors Affecting The Incorporation To College Town For Older Bauta

La presente investigación de tipo explicativa tiene como objetivo caracterizar los factores psicológicos que influyen en la matrícula de Adultos Mayores en la Cátedra Universitaria. La muestra estuvo compuesta por 60 ancianos, de ellos 30 matriculados en la Cátedra y 30 no matriculados, seleccionados a través de una técnica no probalística de muestreo intencional y de participación voluntaria de adultos mayores, residentes en el municipio Bauta. Los instrumentos aplicados son la Escala Psicoafectiva, el test de Yesavage y la Escala Dembo-Rubinstein para determinar los factores psicológicos. El análisis y procesamiento de datos se realiza por el Estadístico de Fisher. Los factores psicológicos que influyen en la matrícula son: Autoestima, proyecto de vida, autovaloración adecuada y la motivación hacia el estudio.

The present research of explanatory type has the purpose of characterizing the psychological facts which affect the registration of the elderly in the Universitary Chair. The model was composed of 60 elders; out of them, 60 registered in the Chair and 30 not registered, selected through a non probabilistic technique of intentional selection and volunteer participation of elders residents of Bauta municipality. The instruments applied were the Psycho-affective Scale, Yesavage Test and Dembo- Rubinstein Scale, to determine the psychological factors. The analysis and data processing was done by Fisher Statistical. The Psychological facts that affect the registrations are: self-esteem,life project, adequate self valuation, and motivation toward the study.

Single-port laparoscopic pelvic and para-aortic lymph node sampling or lymphadenectomy: development of a technique and instrumentation.

BACKGROUND AND OBJECTIVES: Innovations in minimally invasive surgery have allowed surgeons to perform increasingly complex surgeries through smaller incisions. We describe the feasibility and the technique of single-port laparoscopic pelvic and para-aortic lymph node sampling or lymphadenectomy in gynecologic malignancies. METHODS: The study was approved by the institutional review board at the Cleveland Clinic (Cleveland, Ohio). Inclusion criterion was patients with apparent early-stage gynecologic malignancies who required pelvic and/or para-aortic lymph node sampling or lymphadenectomy and were candidates for single-port laparoscopy. Procedures were performed through a single 2.0- to 3.0-cm umbilical incision using a single-port device, deflecting-tip laparoscope, and multifunctional instrumentation. RESULTS: Twenty-one patients underwent single-port surgery/staging performed during the study period. The median patient age was 58 years (range, 17-80 years), and the median patient body mass index was 30 mg/kg² (range, 19-46 mg/kg²). Median overall operating time was 120 minutes (range, 60-185 minutes). Median pelvic and para-aortic node counts were 14 (range, 7-19) and 6 (range, 2-14), respectively. CONCLUSIONS: In this preliminary report, the technique was feasible, and no morbidity was noted. Further studies are needed to better define the ideal gynecologic oncology procedures for single-site surgery and to assess the relative benefits of this new technique compared with more conventional minimally invasive approaches.