Effectiveness and toxicity of five-fraction prone accelerated partial breast irradiation.

PURPOSE: Our institution was an early adopter of 5-fraction accelerated partial breast irradiation (ABPI) to treat women with early-stage breast cancer. This study reports long-term oncologic and cosmetic outcomes. METHODS: We included patients receiving APBI 600 cGy × 5 fx delivered every other day or every day between 2010 and 2022. Logistic regression models were used to identify factors associated with development of late toxicities, clinician, and patient-rated cosmesis. Kaplan-Meier methodology was used to calculate overall survival (OS), disease-free survival (DFS), and locoregional recurrence-free survival (LR-RFS). RESULTS: 442 patients received APBI either daily (56%) or every other day (44%) in the prone position (92%). At a median follow-up of 48 months (range: 5.96-155 months), 12 (2.7%) patients developed a local recurrence (LR). Out of 258 patients with > 3-month toxicity data available, the most common late grade ≥ 2 adverse event was breast fibrosis (6.2%). On multivariate analysis, daily APBI treatment (vs every other day) did not correlate with an increased risk of any late grade ≥ 2 toxicity though it did correlate with a lower risk of any late grade ≥ 2 fibrosis. Overall, at a median follow-up of 80 months, the rates of good-excellent physician and patient-rated cosmesis were 95% and 85%, respectively, with no difference between patients treated on consecutive vs. every other day. On multivariate analysis, patients who did not receive any adjuvant therapy were at increased risk of developing a LR. Five-year OS, LRFS, and DFS were 97.2%, 97.7%, and 89.5%, respectively. CONCLUSIONS: Five-fraction APBI delivered primarily in the prone position either daily or every other day was effective with low rates of local recurrence, minimal toxicity, and excellent cosmesis at long-term follow-up.

Risk of Radiation Dermatitis in Patients With Skin of Color Who Undergo Radiation to the Breast or Chest Wall With and Without Regional Nodal Irradiation.

PURPOSE: Acute radiation dermatitis (ARD) is common after radiation therapy for breast cancer, with data indicating that ARD may disproportionately affect Black or African American (AA) patients. We evaluated the effect of skin of color (SOC) on physician-reported ARD in patients treated with radiation therapy. METHODS AND MATERIALS: We identified patients treated with whole breast or chest wall ± regional nodal irradiation or high tangents using 50 Gy in 25 fractions from 2015 to 2018. Baseline skin pigmentation was assessed using the Fitzpatrick scale (I = light/pale white to VI = black/very dark brown) with SOC defined as Fitzpatrick scale IV to VI. We evaluated associations among SOC, physician-reported ARD, late hyperpigmentation, and use of oral and topical treatments for RD using multivariable models. RESULTS: A total of 325 patients met eligibility, of which 40% had SOC (n = 129). On multivariable analysis, Black/AA race and chest wall irradiation had a lower odds of physician-reported grade 2 or 3 ARD (odds ratio [OR], 0.110; 95% confidence interval [CI], 0.030-0.397; P = .001; OR, 0.377; 95% CI, 0.161-0.883; P = .025), whereas skin bolus (OR, 8.029; 95% CI, 3.655-17.635; P = 0) and planning target volume D0.03cc (OR, 1.001; 95% CI, 1.000-1.001; P = .028) were associated with increased odds. On multivariable analysis, SOC (OR, 3.658; 95% CI, 1.236-10.830; P = .019) and skin bolus (OR, 26.786; 95% CI, 4.235-169.432; P = 0) were associated with increased odds of physician-reported late grade 2 or 3 hyperpigmentation. There was less frequent use of topical steroids to treat ARD and more frequent use of oral analgesics in SOC versus non-SOC patients (43% vs 63%, P < .001; 50% vs 38%, P = .05, respectively). CONCLUSIONS: Black/AA patients exhibited lower odds of physician-reported ARD. However, we found higher odds of late hyperpigmentation in SOC patients, independent of self-reported race. These findings suggest that ARD may be underdiagnosed in SOC when using the physician-rated scale despite this late evidence of radiation-induced skin toxicity.

Early effectiveness and toxicity outcomes of reirradiation after breast conserving surgery for recurrent or new primary breast cancer.

PURPOSE: Breast reirradiation (reRT) after breast conserving surgery (BCS) has emerged as a viable alternative to mastectomy for women presenting with recurrent or new primary breast cancer. There are limited data on safety of different fractionation regimens. This study reports safety and efficacy among women treated with repeat BCS and reRT. METHODS AND MATERIALS: Patients who underwent repeat BCS followed by RT from 2015 to 2021 at 2 institutions were analyzed. Univariate logistic regression models were used to identify predictors of acute and late toxicities. Kaplan-Meier estimates were used to evaluate overall survival (OS), distant metastasis-free survival (DMFS) and locoregional recurrence-free survival (LR-RFS). RESULTS: Sixty-six patients were reviewed with median follow-up of 16 months (range: 3-60 months). At time of first recurrence, 41% had invasive carcinoma with a ductal carcinoma in situ (DCIS) component, 41% had invasive carcinoma alone and 18% had DCIS alone. All were clinically node negative. For the reirradiation course, 95% received partial breast irradiation (PBI) (57.5% with 1.5 Gy BID; 27% with 1.8 Gy daily; 10.5% with hypofractionation), and 5% received whole breast irradiation (1.8-2 Gy/fx), all of whom had received PBI for initial course. One patient experienced grade 3 fibrosis, and one patient experienced grade 3 telangiectasia. None had grade 4 or higher late adverse events. We found no association between the fractionation of the second course of RT or the cumulative dose (measured as EQD2) with acute or late toxicity. At 2 years, OS was 100%, DMFS was 91.6%, and LR-RFS was 100%. CONCLUSION: In this series of patients with recurrent or new primary breast cancer, a second breast conservation surgery followed by reirradiation was effective with no local recurrences and an acceptable toxicity profile across a range of available fractionation regimens at a median follow up of 16 months. Longer follow up is required.

Definitive Radiation With Nodal Boost for Patients With Locally Advanced Breast Cancer.

PURPOSE: The optimal local therapy of patients with nodal disease in supraclavicular (SCV), internal mammary nodes (IMN) and level III axilla is not well studied. We aimed to evaluate the outcomes of patients with breast cancer and advanced nodal disease that received a nodal boost. METHODS AND MATERIALS: This retrospective study included 79 patients with advanced nodal disease who underwent adjuvant radiation with a nodal boost to the SCV, IMNs, and/or axilla. All patients had radiographic changes after systemic therapy concerning for gross nodal disease. Overall survival, disease-free survival (DFS), and local recurrence-free survival were estimated using the Kaplan-Meier method. RESULTS: All patients received an initial 50 Gy to the breast/chest wall and regional nodes, of whom 46.8% received an IMN boost, 38.0% axillary (ax)/SCV boost, and 15.2% both IMN and ax/SCV boost (IMN + ax/SCV). Most patients had hormone receptor positive (74.7%) and human epidermal growth factor receptor 2 negative disease (83.5%). In addition, 12.7% of patients had clinical (c) N2 disease, 21.5% cN3A disease, 51.9% cN3B disease, and 5.1% cN3C disease. Most patients received chemotherapy (97.5%). The median nodal boost dose was 10 Gy (range, 10-20 Gy), with 21.6% of IMN, 16.7% of ax/SCV, and 16.7% of IMN + ax/SCV receiving 14 to 20 Gy. With a median follow up of 30 months, the 3-year local recurrence-free survival, DFS, and overall survival rates were 94.5%, 86.3%, and 93.8%, respectively. Crude rates of failure were 13.9% (10.1% distant failure [DF] alone; 3.8% DF + locoregional failure [LRF]). Rates of failure by boost group were 13.3% for ax/SCV (10.0% DF alone; 3.3% DF + LRF), 5.4% for IMN (2.7% DF alone, 2.7% DF + LRF), and 41.7% for IMN + ax/SCV (33.3% DF, 8.3% DF + LRF). There were no LRFs without DFs. The median time to failure was 22.8 months (interquartile range, 18-34 months). Clinical tumor size and IMN + ax/SCV versus IMN or ax/SCV alone was associated with worse DFS (hazard ratio [HR]: 9.78; 95% confidence interval [CI], 2.07-46.2; P = .004 and HR: 9.49; 95% CI, 2.67-33.7; P = .001, respectively). On multivariate analysis, IMN + ax/SCV versus IMN or ax/SCV alone retained significance (HR: 4.80; 95% CI, 1.27-18.13; P = .02). CONCLUSIONS: In this population of patients with locally advanced breast cancer, the majority of failures were distant with no isolated LRFs. Failures were the highest in the IMN + ax/SCV group (∼40%). Further treatment escalation is necessary for these patients.

Disparities in the Uptake of Telemedicine and Implications for Clinical Trial Enrollment in Patients With Breast Cancer.

PURPOSE: Since the COVID-19 pandemic, telemedicine has emerged as an alternative to office visits in routine radiation oncology practice. The purpose of this study was to identify factors associated with patient preference for an initial consult via telemedicine and correlation with clinical trial enrollment. METHODS AND MATERIALS: We evaluated patients with breast cancer seen during the open enrollment of a prospective randomized trial from June 1, 2020, to May 13, 2021. Univariate and multivariate logistic regression models were used to identify factors associated with virtual versus in-person initial consultation. All statistical tests were 2-sided, and the null hypothesis was rejected for P < .05. RESULTS: We identified 476 patient consultations with 259 office visits and 217 telemedicine visits. On multivariate analysis, increased age, unemployment, chemotherapy receipt, and radiation at our institution were associated with decreased usage of telemedicine for consultation visit. Out of 217 patients who underwent a telemedicine initial consultation, 10% were eligible to enroll on the trial, and of those eligible 76% enrolled. Out of 259 patients who underwent office visit initial consultation, 14% were eligible to enroll on the trial, and of those eligible 53% enrolled. Among eligible patients, there was no statistically significant difference in clinical trial enrollment between telemedicine and office visits. CONCLUSIONS: Older patients, unemployed patients, those receiving chemotherapy, and those who subsequently received radiation at our institution were less likely to use telemedicine for their initial consult. Despite these disparities in telemedicine usage, there was no difference in clinical trial enrollment. Telemedicine may be an effective platform for clinical trial enrollment though further strategies to improve its access are essential.

A rapid antibody screening haemagglutination test for predicting immunity to SARS-CoV-2 variants of concern.

Background: Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection. Methods: Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80-99 years, n = 89) and younger adults (23-77 years, n = 310) and compared to convalescent sera from naturally infected individuals (1-89 years, n = 307). Results: Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72-0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72-76%) older adults respond after two vaccinations to alpha and delta, but only 58-62% to beta and gamma, compared to 96-97% of younger vaccinees and 68-76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination. Conclusions: Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness.

Five-Fraction Prone Accelerated Partial Breast Irradiation: Long-Term Oncologic, Dosimetric, and Cosmetic Outcome.

PURPOSE: Randomized data support accelerated partial breast irradiation (APBI) for early-stage breast cancer with variable techniques and cosmesis outcomes. We have treated patients with 5-fraction prone external beam APBI for over a decade and herein report acute and late outcomes. METHODS AND MATERIALS: Patients receiving APBI 600 cGy × 5 between 2010 and 2019 were included. APBI was primarily delivered prone, with opposed tangents targeting the tumor bed expanded by 1.5 cm (cropped 6 mm from skin). Ipsilateral breast was constrained to V50% < 60% and V100% < 35%. Survival was estimated with Kaplan-Meier. Late toxicities and clinician- and patient-rated cosmesis were evaluated for patients with >6 months follow-up (FU). RESULTS: Of 345 patients meeting criteria, 14 were excluded due to APBI given for ipsilateral breast tumor recurrence (IBTR; n = 3), palliation (n = 9), and incomplete radiation therapy course (n = 2). Of the 331 remaining, median age was 70, 7.2% had ductal carcinoma in situ, and 94.3% were treated prone, with 32% treated every other day and 68% on consecutive days. Mean heart dose was 23.8 cGy for left-sided and 12.7 cGy for right-sided cancers. Ipsilateral lung V30% was 0.4%. At 5-year median FU, there were 7 (2.1%) IBTR, 9 (2.7%) contralateral recurrences, and 1 (0.3%) distant metastasis. Five-year local recurrence-free, disease-free, and overall survival was 99.5%, 96.7%, and 98.1%, respectively. When comparing patients with IBTR versus without, a higher proportion did not receive hormone therapy (71.4% vs. 26.2%, P = .018). Rates of acute grade 1 to 2 dermatitis, fatigue, and pain were 35.4%, 21.8%, and 9.4%, respectively, with no grade 3 toxicity. The rate of good-excellent physician- and patient-rated cosmesis (n = 199, median FU 2.8 years) was 92.5% and 89.4%, respectively. Patients experienced low rates of telangiectasia, fibrosis, and retraction/atrophy. CONCLUSIONS: We report excellent dosimetric, oncologic, cosmetic, and late toxicity outcomes for patients treated with 5-fraction APBI. To our knowledge this is the largest series of women treated with prone APBI.

Effects of Severe Acute Respiratory Syndrome Coronavirus 2 Strain Variation on Virus Neutralization Titers: Therapeutic Use of Convalescent Plasma.

We compared neutralizing antibody titers of convalescent samples collected before and after the emergence of novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), against the wild-type virus and Alpha (B.1.1.7) and Beta (B.1.351) variants. Plasma samples collected in 2020 before emergence of variants showed reduced titers against the Alpha variants, and both sets of samples demonstrated significantly reduced titers against Beta. Comparison of microneutralization titers with those obtained with pseudotype and hemagglutination tests showed a good correlation between their titers and effects of strain variation, supporting the use of these simpler assays for assessing the potency of convalescent plasma against currently circulating and emerging strains of SARS-CoV-2.

Toxicity and Cosmetic Outcome of Breast Irradiation in Women with Breast Cancer and Autoimmune Connective Tissue Disease: The Role of Fraction and Field Size.

PURPOSE: Hypofractionation has historically been underused among breast cancer patients with connective tissue diseases given a theoretical risk of increased toxicity and their overall underrepresentation in clinical trials that established hypofractionation as standard of care. We aim to compare the rates of toxicity in patients with autoimmune connective tissue diseases treated with conventionally fractioned radiation therapy (CF-RT) and hypofractionated radiation therapy (HF-RT) including accelerated partial breast irradiation. METHODS: A total of 1983 patients treated with breast conservation between 2012 and 2016 were reviewed for diagnosis of autoimmune disease. Univariate analysis using binary logistic regression was performed to evaluate the effect of disease and treatment variables on acute and late toxicity. Multivariate analyses using Cox regression models were used to evaluate the independent associations between covariates and the primary end points. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for reach risk group. RESULTS: Ninety-two patients with autoimmune disease were identified. Median follow-up was 59 months. Of the patients 35% received CF-RT and 65% received HF-RT, of whom 70% received whole breast radiation (WBI) without regional nodal irradiation, 12% received WBI with regional nodal irradiation, and 18% received accelerated partial breast radiation. Patients who received CF-RT were significantly more likely to have autoimmune disease (AD) symptoms (78% vs 37%, P <.001), to be managed on disease-modifying antirheumatic drugs (DMARDs; 41% vs 15%, P = .013), and to have active autoimmune disease (84% vs 43%, P <.001). On multivariate analysis, HF-RT was associated with a significantly decreased odds of acute and late grade 2/3 toxicity compared with CF-RT fractionation (acute: OR 0.200, 95% CI 0.064-0.622, P = .005; late: OR 0.127, 95% CI 0.031-0.546, P = .005). CONCLUSIONS: Hypofractionation including accelerated partial-breast irradiation is associated with less acute or late grade 2/3 toxicity in this population.

Seroprevalence of SARS-CoV-2 Infection in the Colombo Municipality Region, Sri Lanka.

Background: As the Municipality Council area in Colombo (CMC) experienced the highest number of cases until the end of January 2021, in Sri Lanka, we carried out a serosurvey prior to initiation of the vaccination program to understand the extent of the SARS-CoV-2 outbreak. Methods: SARS-CoV-2 seropositivity was determined in 2,547 individuals between the ages of 10-86 years, by the Wantai total antibody ELISA. We also compared seroprevalence using the haemagglutination test (HAT) to evaluate its usefulness in carrying out serosurveys. Results: The overall seropositivity rate was 24.46%, while seropositivity by HAT was 18.90%. Although The SARS-CoV-2 infection detection rates by PCR were highest in the population between the ages of 20-60 years of age, there was no statistically significant difference in the seropositivity rates in different age groups. For instance, although the seropositivity rate was highest in the 10-20 age group (34.03%), the PCR positivity rate was 9.80%. Differences in the PCR positivity rates and seropositivity rates were also seen in 60-70-year-olds (8.90 vs. 30.4%) and in individuals >70 years (4.10 vs. 1.20%). The seropositivity rate of the females was 29.70% (290/976), which was significantly higher (p < 0.002) than in males 21.2% (333/1,571). Conclusions: A high seroprevalence rate (24.5%) was seen in all age groups in the CMC suggesting that a high level of transmission was seen during this time. The higher PCR positivity rates between the ages of 20-60 are likely to be due to increased testing carried out in the working population. Therefore, the PCR positivity rates, appear to underestimate the true extent of the outbreak and the age groups which were infected.

Comparability of six different immunoassays measuring SARS-CoV-2 antibodies with neutralizing antibody levels in convalescent plasma: From utility to prediction.

BACKGROUND: Convalescent plasma (CP) therapy for coronavirus disease (COVID-19) provides virus-neutralizing antibodies that may ameliorate the outcome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. The effectiveness of CP likely depends on its antiviral neutralizing potency and is determined using in vitro neutralizing antibody assays. STUDY DESIGN AND METHODS: We evaluated abilities of three immunoassays for anti-spike antibodies (EUROimmun, Ortho, Roche), a pseudotype-based neutralization assay, and two assays that quantify ACE2 binding of spike protein (GenScript and hemagglutination test [HAT]-based assay) to predict neutralizing antibody titers in 113 CP donations. Assay outputs were analyzed through linear regression and calculation of sensitivities and specificities by receiver operator characteristic (ROC) analysis. RESULTS: Median values of plasma samples containing neutralizing antibodies produced conversion factors for assay unitage of ×6.5 (pseudotype), ×19 (GenScript), ×3.4 (HAT assay), ×0.08 (EUROimmun), ×1.64 (Roche), and ×0.10 (Ortho). All selected assays were sufficient in identifying the high titer donations based on ROC analysis; area over curve ranged from 91.7% for HAT and GenScript assay to 95.6% for pseudotype assay. However, their ability to predict the actual neutralizing antibody levels varied substantially as shown by linear regression correlation values (from 0.27 for Ortho to 0.61 for pseudotype assay). DISCUSSION: Overall, the study data demonstrate that all selected assays were effective in identifying donations with high neutralizing antibody levels and are potentially suitable as surrogate assays for donation selection for CP therapy.

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2.

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world.

Breast Conservation in Women with Autoimmune Disease: The Role of Active Autoimmune Disease and Hypofractionation on Acute and Late Toxicity in a Case-Controlled Series.

PURPOSE: Autoimmune connective tissue disease (CTD) has historically represented a relative contraindication to breast conservation (BC) among patients with early-stage breast cancer. Controversy exists regarding the use of hypofractionated radiation therapy (RT) among patients with CTDs. We evaluated acute and late toxicity in patients with breast cancer and CTD treated with BC. METHODS AND MATERIALS: Of 1983 patients treated with BC from 2012 to 2016, we identified 91 patients with an autoimmune disease (AD). Each patient was matched to a control without AD based on age, RT field, and fractionation. RT toxicity and clinician-rated cosmesis were compared between cases and controls. Overall survival, disease-free survival, and local recurrence-free survival were estimated using the Kaplan-Meier method. RESULTS: The median follow-up was 49.9 months for cases and 53.0 months for controls, and 67% of cases and controls were treated with hypofractionated RT. There was no difference in grade 2/3 acute toxicity between cases and controls (26.4% vs. 16.5%, respectively; P = .148). There was a significantly higher rate of grade 2/3 late toxicity among cases (25.8% vs 12.1% among controls; P = .049). Active AD at the time of RT increased the rate of grade 2/3 late toxicity compared with controls (41.7% in cases vs. 11.4% in controls; P = .018). Among patients treated with hypofractionated RT, there was no difference in acute or late grade 2/3 toxicity between cases and controls (acute: 13.1% in cases vs. 11.5% in controls [P > 0.9]; late: 11.9% in cases vs. 13.1% in controls [P > 0.9]). The rates of good/excellent clinician-rated cosmesis were similar between groups (92.9% in cases vs. 98.9% in controls; P = .142). CONCLUSIONS: In the largest matched case-control study of patients with CTD treated with conventional and hypofractionated RT, we demonstrate low rates of radiation toxicity, with good to excellent clinician-rated cosmesis. There was increased late toxicity in cases, especially in patients with active AD at time of RT. There was no increase in acute or late toxicity in the patients treated with hypofractionation.

Modern Management of High-risk Soft Tissue Sarcoma With Neoadjuvant Chemoradiation: A Single-center Experience.

OBJECTIVE: Neoadjuvant chemoradiation (NA-CRT), followed by resection of high-risk soft tissue sarcoma (STS), may offer good disease control and toxicity outcomes. We report on a single institution's modern NA-CRT experience. MATERIALS AND METHODS: Delay to surgical resection, resection margin status, extent of necrosis, tumor cell viability, presence of hyalinization, positron emission tomography (PET)/computed tomography data, and treatment toxicities were collected. Using the Kaplan-Meier survival analysis, 5-year overall survival, disease-free survival, distant metastasis-free survival, and local control (LC) were estimated. Clinicopathologic features and PET/computed tomography avidity changes were assessed for their potential predictive impact using the log-rank test. RESULTS: From 2011 to 2018, 37 consecutive cases of localized high-risk STS were identified. Twenty-nine patients underwent ifosfamide-based NA-CRT to a median dose of 50 Gy before en bloc resection. At a median follow-up of 40.3 months, estimated 5-year overall survival was 86.1%, disease-free survival 70.2%, distant metastasis-free survival 75.2%, and LC 86.7%. Following NA-CRT, a median reduction of 54.7% was observed in tumor PET avidity; once resected, median tumor necrosis of 60.0% with no viable tumor cells was detected in 13.8% of the cases. Posttreatment resection margins were negative in all patients, with 27.6% having a margin of ≤1 mm. Delays of over 6 weeks following the end of radiation treatment to surgical resection occurred in 20.7% cases and was suggestive of inferior LC (92.8% vs. 68.6%, P=0.025). CONCLUSIONS: This single-institution series of NA-CRT demonstrates favorable disease control. Delay in surgical resection was associated with inferior LC, a finding that deserves further evaluation in a larger cohort. LEVEL OF EVIDENCE: Level III-retrospective cohort study.

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions.

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.

Therapeutic Monoclonal Antibodies for Ebola Virus Infection Derived from Vaccinated Humans.

We describe therapeutic monoclonal antibodies isolated from human volunteers vaccinated with recombinant adenovirus expressing Ebola virus glycoprotein (EBOV GP) and boosted with modified vaccinia virus Ankara. Among 82 antibodies isolated from peripheral blood B cells, almost half neutralized GP pseudotyped influenza virus. The antibody response was diverse in gene usage and epitope recognition. Although close to germline in sequence, neutralizing antibodies with binding affinities in the nano- to pico-molar range, similar to "affinity matured" antibodies from convalescent donors, were found. They recognized the mucin-like domain, glycan cap, receptor binding region, and the base of the glycoprotein. A cross-reactive cocktail of four antibodies, targeting the latter three non-overlapping epitopes, given on day 3 of EBOV infection, completely protected guinea pigs. This study highlights the value of experimental vaccine trials as a rich source of therapeutic human monoclonal antibodies.

Characterization of Influenza Virus Pseudotyped with Ebolavirus Glycoprotein.

We have produced a new Ebola virus pseudotype, E-S-FLU, that can be handled in biosafety level 1/2 containment for laboratory analysis. The E-S-FLU virus is a single-cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza virus hemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU virus production. Infection of cells with the E-S-FLU virus was dependent on the Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. The E-S-FLU virus was neutralized specifically by an anti-Ebolavirus glycoprotein antibody and a variety of small drug molecules that are known to inhibit the entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC1280; Sigma) of 1,280 pharmacologically active compounds for inhibition of virus entry. A total of 215 compounds inhibited E-S-FLU virus infection, while only 22 inhibited the control H5-S-FLU virus coated in H5 hemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action, e.g., calcium channel blockers, estrogen receptor antagonists, antihistamines, serotonin uptake inhibitors, etc., and this correlates with inhibitor screening results obtained with other pseudotypes or wild-type Ebola virus in the literature. The E-S-FLU virus is a new tool for Ebola virus cell entry studies and is easily applied to high-throughput screening assays for small-molecule inhibitors or antibodies.IMPORTANCE Ebola virus is in the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates for Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus and screen for inhibitors. Here we characterized a new surrogate, named E-S-FLU virus, that is based on a disabled influenza virus core coated with the Ebola virus surface protein but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU virus uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU virus in biosafety level 1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small-molecule library of 1,280 candidates for inhibitors of viral entry.

Expression of High Mobility Group Protein B1 in Cardiac Tissue of Elderly Patients with Coronary Artery Disease with or without Inflammatory Rheumatic Disease.

BACKGROUND: It is known from clinical practice and observational studies that elderly patients with a diagnosis of inflammatory rheumatic diseases (IRD) bear a significantly increased risk for cardiovascular diseases such as coronary artery disease (CAD) and heart failure. The molecular mechanism, however, is still not known. Recently, high mobility group protein B1 (HMGB1), a ubiquitous, highly conserved single polypeptide expressed in all mammal eukaryotic cells, has been identified to mediate myocardial dysfunction in vitro once released from the nuclei of cardiomyocytes. OBJECTIVE: To investigate whether HMGB1 and its receptors are expressed in cardiac muscles of elderly patients with CAD with or without IRD. METHODS: HMGB1 and its 3 well-known receptors, receptor for advanced glycation end products, Toll-like receptor 2 (TLR2), and TLR4, were examined by immunohistochemistry on myocardial biopsy specimens from 18 elderly patients with CAD (10 with IRD, 8 without IRD). Furthermore, total HMGB1 protein levels were measured by Western blot from the cardiac biopsies in 5 patients with and 5 without IRD. RESULTS: Pathologic cytosolic HMGB1 in cardiomyocytes was massively recorded in all patients with IRD, but only slightly expressed in 1 patient without IRD. Total HMGB1 levels were also consistently lower in myocardial muscle biopsies of patients with IRD compared to those without IRD. Furthermore, all 3 HMGB1 receptors were expressed in cardiomyocytes of all patients. CONCLUSION: The increased cytosolic expression of HMGB1 in cardiomyocytes and the lower total amount of HMGB1 in the cardiac specimens of IRD patients is consistent with a greater release of HMGB1 from the myocardial nuclei in IRD than non-IRD individuals. Thus, the HMGB1 signaling pathways may be more easily activated in elderly CAD patients with concomitant IRD and trigger a detrimental inflammatory process causing severe cardiovascular problems. Therefore, targeting HMGB1 in IRD patients might reduce the risk for cardiovascular events.