Defining the dimensions of circulating tumor cells in a large series of breast,prostate, colon,and bladder cancer patients

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient‐derived CTCs acquired on cartridges of the FDA‐cleared CellSearch^® method were retrospectively collected and automatically re‐analyzed using the accept software package. The median CTC diameter (μm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch^® cartridges containing 71 612 CTCs were included. In BC, the median computed diameter (CD) of patient‐derived CTCs was 12.4 μm vs 18.4 μm for cultured cell line cells. For PC, CDs were 10.3 μm for CTCs vs 20.7 μm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5 μm and 8.6 μm, respectively. Finally, leukocytes were 9.4 μm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size‐based isolation methods.

Abbreviations

ACCEPT

Automated CTC Classification, Enumeration, and PhenoTyping software

BC

breast cancer

BLC

bladder cancer

CD

computed diameter

CEL

cultured tumor cell (cell line)

CK

cytokeratin

CRC

colorectal cancer

CTC‐L

circulating tumor cells derived from cerebrospinal fluid (liquor)

CTCs

circulating tumor cells

DAPI

4′6‐diamidino‐2‐phenylindole

EMT

epithelial–mesenchymal transition

EpCAM

epithelial cell adhesion molecule

IQR

interquartile range

KW test

Kruskal–Wallis test

MWU test

Mann–Whitney U test

NCR

nucleus/cytoplasm ratio

P2A

perimeter to area

PC

prostate cancer

TIF

tagged Image Format files

TXT

text file

μm

micrometer

µm²

square micrometers

     

Predicting osimertinib‐treatment outcomes through EGFR mutant‐fraction monitoring in the circulating tumor DNA of EGFR T790M‐positive patients with non‐small cell lung cancer (WJOG8815L)

The WJOG8815L phase II clinical study involves patients with non‐small cell lung cancer (NSCLC) that harbored the EGFR T790M mutation, which confers resistance to EGFR tyrosine kinase inhibitors (TKIs). The purpose of this study was to assess the predictive value of monitoring EGFR genomic alterations in circulating tumor DNA (ctDNA) from patients with NSCLC that undergo treatment with the third‐generation EGFR‐TKI osimertinib. Plasma samples of 52 patients harboring the EGFR T790M mutation were obtained pretreatment (Pre), on day 1 of treatment cycle 4 (C4) or cycle 9 (C9), and at diagnosis of disease progression or treatment discontinuation (PD/stop). CtDNA was screened for EGFR‐TKI‐sensitizing mutations, the EGFR T790M mutation, and other genomic alterations using the cobas EGFR Mutation Test v2 (cobas), droplet digital PCR (ddPCR), and targeted deep sequencing. Analysis of the sensitizing—and T790M—EGFR mutant fractions (MFs) was used to determine tumor mutational burden. Both MFs were found to decrease during treatment, whereas rebound of the sensitizing EGFR MF was observed at PD/stop, suggesting that osimertinib targeted both T790M mutation‐positive tumors and tumors with sensitizing EGFR mutations. Significant differences in the response rates and progression‐free survival were observed between the sensitizing EGFR MF‐high and sensitizing EGFR MF‐low groups (cutoff: median) at C4. In conclusion, ctDNA monitoring for sensitizing EGFR mutations at C4 is suitable for predicting the treatment outcomes in NSCLC patients receiving osimertinib (Clinical Trial Registration No.: UMIN000022076).

Abbreviations

CIs

confidence intervals

ctDNA

circulating tumor DNA

ddPCR

droplet digital PCR

EGFR

epidermal growth factor receptor

MFs

mutant fractions

NGS

next‐generation sequencing

NSCLC

non‐small cell lung cancer

ORR

overall response rate

OS

overall survival

PD

progressive disease

PFS

progression‐free survival

PR

partial response

SD

stable disease

TKI

tyrosine kinase inhibitor

     

Comparative Outcome Analysis of N-Butyl Cyanoacrylate Embolization of the False Lumen Versus Thoracic Endovascular Aortic Repair in Aortic Dissection

PurposeTo evaluate the feasibility, safety, and effectiveness of N-butyl cyanoacrylate (NBCA) embolization for the treatment of aortic dissection.Materials and MethodsIn this single-center retrospective study conducted from February 2003 to June 2019, NBCA embolization of an aortic false lumen was attempted in 12 patients (median age, 59 y; range, 41–68 y) and thoracic endovascular aortic repair (TEVAR) was performed in 53 patients (median age, 59 y; range, 37–70 y) for aortic dissection with one or more indications of persisting pain, malperfusion, rupture or impending rupture, maximal aortic diameter ≥ 55 mm, and/or rapid aortic enlargement. The main exclusion criterion for embolization was the presence of fast blood flow in the aortic false lumen on aortography. The efficacy of NBCA embolization and TEVAR was compared by evaluating technical and clinical outcomes, repeat intervention–free survival (RFS), and overall survival (OS).ResultsTechnical success was achieved in 11 of the 12 patients treated with NBCA embolization (91.7%), and clinical success was achieved in 9 of these 11 (81.8%). No significant difference was found between embolization and TEVAR in clinical success rates (embolization, 81.8%; TEVAR, 84.9%; P = .409) or procedure-related complications (embolization, 1 patient [8.3%]; TEVAR, 4 patients [7.5%]; P = .701). In addition, embolization showed comparable 5-y RFS (embolization, 82.5% ± 9.3; TEVAR, 85.5% ± 4.8; P = .641) and 5-y OS (embolization, 100%; TEVAR, 95.4% ± 3.2; P = .744) rates to TEVAR.ConclusionsNBCA embolization of the false lumen in aortic dissection seems to be a safe and effective treatment modality for the closure of false lumen in selected patients.     

Digital surface scanning in flap perfusion

Monitoring vascular perfusion of transferred tissue is essential in reconstructive surgery to recognize early flap failure. The aim of this study was to evaluate the ability of a digital surface scanner to detect vascular perfusion disorders through the monitoring of skin colour changes. A total of 160 surface scans of the forearm skin were performed with a TRIOS 3D scanner. Vascular compromise was simulated at different time-points by intermittent occlusion of the blood supply to the forearm skin (first the arterial blood supply and then the venous blood supply). Skin colour changes were examined according to the hue, saturation, and value colour scale. Colour differences were analysed with a paired t-test. Significant differences were observed between the colour of the normal skin and that of the vascular compromised skin (P < 0.01). The surface scanner could distinguish between arterial occlusion and venous congestion (P < 0.01). A digital surface scan is an objective, non-invasive tool to detect early vascular perfusion disorders of the skin.     

Efficacy of recombinant Marek’s disease virus vectored vaccines with computationally optimized broadly reactive antigen (COBRA) hemagglutinin insert against genetically diverse H5 high pathogenicity avian influenza viruses

The genetic and antigenic drift associated with the high pathogenicity avian influenza (HPAI) viruses of Goose/Guangdong (Gs/GD) lineage and the emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. Here, we tested experimental vector herpesvirus of turkey (vHVT)-H5 vaccines containing either wild-type clade 2.3.4.4A-derived H5 inserts or computationally optimized broadly reactive antigen (COBRA) inserts with challenge by homologous and genetically divergent H5 HPAI Gs/GD lineage viruses in chickens. Direct assessment of protection was confirmed for all the tested constructs, which provided clinical protection against the homologous and heterologous H5 HPAI Gs/GD challenge viruses and significantly decreased oropharyngeal shedding titers compared to the sham vaccine. The cross reactivity was assessed by hemagglutinin inhibition (HI) and focus reduction assay against a panel of phylogenetically and antigenically diverse H5 strains. The COBRA-derived H5 inserts elicited antibody responses against antigenically diverse strains, while the wild-type-derived H5 vaccines elicited protection mostly against close antigenically related clades 2.3.4.4A and 2.3.4.4D viruses. In conclusion, the HVT vector, a widely used replicating vaccine platform in poultry, with H5 insert provides clinical protection and significant reduction of viral shedding against homologous and heterologous challenge. In addition, the COBRA-derived inserts have the potential to be used against antigenically distinct co-circulating viruses and future drift variants.     

Pulmonary metastases in urogenital cancers: Surgical treatment and outcomes

IntroductionMetastasis is remaining one of the major problems in cancer treatment. Like many other malignancies, urogenital tumors originating from kidney, prostate, testes, and bladder tend to metastasize to the lungs.The aim of this retrospective study is to evaluate the operative results and prognosis of pulmonary metastasectomy in patients with primary urogenital tumors.MethodsThis study was approved by the local ethical committee. We retrospectively analyzed the surgical and oncological results of patients who underwent lung resections for urogenital cancer metastases in our department between 2002 and 2018. Demographic data and clinicopathological features were extracted from the medical records. Survival outcomes according to cancer subtypes and early postoperative results of VATS and thoracotomy were analyzed.Results22 out of 126 patients referred for pulmonary metastasectomy to our department had metastases from urogenital tumors. These patients consisted of 17 males and five females. Their metastasis originated from renal cell carcinoma (RCC; n = 9), bladder tumor (n = 7), testis tumors (n = 4), and prostate cancer (n = 2). There was no intraoperative complication. Postoperative complications were seen in 2 patients.ConclusionsAlthough pulmonary metastasectomy in various types of tumors is well known and documented, the data is limited for metastases of urogenital cancers in the literature. Despite the limitations of this study, we aim to document our promising results of pulmonary metastasectomy in patients with primary urogenital tumors and wanted to emphasize the role of minimally invasive approaches.     

形觉剥夺性高度近视豚鼠巩膜形态改变及缺氧诱导因子-1α和氧自由基在高度近视中的作用

目的　观察形觉剥夺性高度近视（form deprivation high myopia，FDHM）豚鼠巩膜形态变化，探讨缺氧诱导因子-1α（hypoxia-inducible factor-1α，HIF-1α）及氧自由基在高度近视中的作用。方法　将豚鼠适应性饲养1周后，随机分为空白对照组（25只）和模型组（25只）。模型组豚鼠右眼行眼睑缝合，所有模型组豚鼠均选择右眼作为FDHM组，对侧眼为自身对照组。空白对照组豚鼠不做任何处理。于造模前及造模后8周采用检影镜测量屈光度，A超进行生物测量。形觉剥夺8周以后处死豚鼠，观察巩膜形态和超微结构的变化，测定巩膜HIF-1α相对表达量，超氧化物歧化酶（superoxide dismutase，SOD）活力及丙二醛（malondialdehyde，MDA）的含量。结果　豚鼠形觉剥夺8周以后，FDHM组屈光度从（+3.59±0.33）D变为（-7.96±0.55）D，明显高于空白对照组（+0.89±0.32）D、自身对照组（-0.55±0.49）D（均为P<0.05）；玻璃体腔深度为（4.12±0.13）mm明显高于空白对照组（3.71±0.23）mm和自身对照组（3.93±0.04）mm（均为P<0.05）；眼轴长度为（8.93±0.22）mm明显长于空白对照组（7.95±0.37）mm和自身对照组（8.01±0.15）mm（均为P<0.05）。巩膜组织明显变薄，细胞外基质增多，成纤维细胞密度降低，胶原纤维平均直径减小。FDHM组巩膜中HIF-1α相对表达量、MDA含量明显高于空白对照组和自身对照组，SOD活力明显低于空白对照组和自身对照组（均为P<0.05）。结论　形觉剥夺8周后，豚鼠FDHM眼近视度数明显增加，玻璃体腔深度增加，眼轴延长，巩膜形态发生病理性变化；HIF-1α、SOD、MDA可能参与了FDHM的形成。     

Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective.The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated.In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 10⁶ cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers.Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 10⁵ cells/mL resulted in a virus titer higher than 10⁷ FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 10⁷ FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca⁺⁺ and Mg⁺⁺. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.