Neoadyuvant Treatment in the Covid Era

Since Coronavirus Disease 2019 (COVID-19) was declared pandemic in March 2020, there have been 545.226.550 cases up to 4 July 2022 (1). Several studies concluded that patients (pts) with cancer are at increased risk of COVID-19 infection, morbidity and mortality. Those undergoing neoadyuvant treatment are at particularly risk of disease progression if chemotherapy or surgery are delayed. Also, is known that a higher NLR (neutrophil to limphocyte ratio) is related to worse outcomes (3). Our hospital is located at the Northwest of Spain and in the last months we noticed a never seen number of infections in cancer population. The aim of this study is to evaluate the severity of COVID19 and its impact on chemotherapy and surgery delay in pts undergoing neoadjuvant chemotherapy breast cancer. METHOD(S): We conducted a ambispective, unicenter, observational study of breast cancer pts, treated with neoadjuvant chemotherapy, between March 2020 and May 2022 at University Hospital A Coruna (Spain). We analyzed type of infection, need of hospitalization, chemotherapy and surgical delay, and its association with tumor type;BRCA germline mutation;clinical stage;treatment;vaccination status;and neutrophils, lymphocytes, and NLR before COVID-19 disease. RESULT(S): During the study period, from 1 March 2020 to 31 May 2022, 183 pts underwent neoadjuvant chemotherapy. A total of 23 (12.5%) pts experienced COVID-19 infection, of which 21 were diagnosed between January and May 2022. The median age was 47,91 years [range 33 - 69 years]. Luminal B HER 2 negative comprised the most common molecular subtype (40.9%), followed by Triple Negative (36.4%), Luminal B HER 2 positive (13.6%), and HER 2 enriched (9.1%). Germline mutations in BRCA account for 13.6% pts. At diagnosis, 4.5%, 72.7%, and 22.7% had stages I, II, and III respectively. Chemotherapy treatments included: paclitaxel followed by AdryamicineCyclophosphamide (AC) (45.4%);carboplatin - paclitaxel - trastuzumab - pertuzumab (18,2%);carboplatin - paclitaxel followed by AC (18,2%);KEYNOTE-756: pembrolizumab/placebo - paclitaxel followed by AC (13.7%);and paclitaxel - trastuzumab - pertuzumab followed by myocet - cyclophosphamide - trastuzumab - pertuzumab (4.5%). The association of G-CSF ocurred in 9 pts (40.9%). 22 pts were fully vaccinated, 8 pts (36.4%) with two doses and 13 pts (59.1%) with three doses. 77.3% pts experienced mild respiratory symptoms with 9.1% hospitalizations. The median duration of delays was 15 days for chemotherapy and 29,58 days for surgery. NLR percentil 25 was associated with COVID-19 type of infection. For those pts with a lower rate, infection was asymptomatic and for those with a higher rate symptoms were moderate (X2= 5,119, p = 0,024). CONCLUSION(S): COVID-19 disease become a high prevalent infection in pts undergoing neoadjuvant breast cancer chemotherapy. Most pts are fully vaccinated and experienced an indolent infection. NLR is an easily measurable and cost-effective parameter that could be useful as a prognostic marker of severity in COVID-19. We will continue to follow-up these pts to see the impact of chemotherapy or surgery delay in pathological complete response and disease-free survival until the congress in December 2022.

The OM-85 bacterial lysate: a new tool against SARS-CoV-2?

The emergence of SARS-CoV-2, a novel coronavirus, caused the global Coronavirus disease of 2019 (COVID-19) pandemic. Because SARS-CoV-2 mutates rapidly, vaccines that induce immune responses against viral components critical for target cell infection strongly mitigate but do not abrogate viral spread, and disease rates remain high worldwide. Complementary treatments are therefore needed to reduce the frequency and/or severity of SARS-CoV-2 infections. OM-85, a standardized lysate of 21 bacterial strains often found in the human airways, has immuno-modulatory properties and is widely used empirically in Europe, South America and Asia for the prophylaxis of recurrent upper airway infections in adults and children, with excellent safety profiles. In vitro studies from our laboratory recently demonstrated that OM-85 inhibits SARS-CoV-2 epithelial cell infection by downregulating SARS-CoV-2 receptor expression, raising the possibility that this bacterial extract might eventually complement the current COVID-19 therapeutic toolkit. Here we discuss how our results and those from other groups are fostering progress in this emerging field of research.

SARS-CoV-2 infection of kidney tissues from severe COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests diverse clinical pathologies involving multiple organs. While the respiratory tract is the primary SARS-CoV-2 target, acute kidney injury is common in COVID-19 patients, displaying as acute tubular necrosis (ATN) resulting from focal epithelial necrosis and eosinophilia, glomerulosclerosis, and autolysis of renal tubular cells. However, whether any renal cells are infected by SARS-CoV-2 and the mechanism involved in the COVID-19 kidney pathology remain unclear. METHODS: Kidney tissues obtained at autopsy from four severe COVID-19 patients and one healthy subject were examined by hematoxylin and eosin staining. Indirect immunofluorescent antibody assay was performed to detect SARS-CoV-2 spike protein S1 and nonstructural protein 8 (NSP8) together with markers of different kidney cell types and immune cells to identify the infected cells. RESULTS: Renal parenchyma showed tissue injury comprised of ATN and glomerulosclerosis. Positive staining of S1 protein was observed in renal parenchymal and tubular epithelial cells. Evidence of viral infection was also observed in innate monocytes/macrophages and NK cells. Positive staining of NSP8, which is essential for viral RNA synthesis and replication, was confirmed in renal parenchymal cells, indicating the presence of active viral replication in the kidney. CONCLUSIONS: In fatal COVID-19 kidneys, there are SARS-CoV-2 infection, minimally infiltrated innate immune cells, and evidence of viral replication, which could contribute to tissue damage in the form of ATN and glomerulosclerosis.

SARS-CoV-2 Infects Endothelial Cells In Vivo and In Vitro.

SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.

Different Neutralization Sensitivity of SARS-CoV-2 Cell-to-Cell and Cell-Free Modes of Infection to Convalescent Sera.

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. These findings suggest that the cell-to-cell mode of SARS-CoV-2 transmission, for which the mechanisms are largely unknown, could be of great importance for treatment and prevention of COVID-19.

Recovery of Synthetic Zika Virus Based on Rio-U1 Isolate Using a Genetically Stable Two Plasmid System and cDNA Amplification.

In 2016, the world experienced the unprecedented Zika epidemic. The ZIKV emerged as a major human pathogen due to its association with the impairment of perinatal development and Guillain-Barré syndrome. The occurrence of these severe cases of Zika points to the significance of studies for understanding the molecular determinants of flavivirus pathogenesis. Reverse genetics is a powerful method for studying the replication and determinants of pathogenesis, virulence, and viral attenuation of flaviviruses, facilitating the design of vaccines and therapeutics. However, the main hurdle in the development of infectious clones is the instability of full-length cDNA in Escherichia coli. Here, we described the development of a genetically stable and efficient infectious clone based on the ZIKV Rio-U1 isolated in the 2016 epidemic in Brazil. The employed strategy consisted of cloning the viral cDNA genome into two stable plasmid subclones and obtaining a high-quality cDNA template with increment in DNA mass for in vitro transcription by PCR amplification. The strategy for developing a ZIKV infectious cDNA clone designed in this study was successful, yielding a replicative and efficient clone-derived virus with high similarities with its parental virus, Rio-U1, by comparison of the proliferation capacity in mammal and insect cells. The infection of AG129 immunocompromised mice caused identical mortality rates, with similar disease progression and morbidity in the animals infected with the parental and the cDNA-derived virus. Histopathological analyses of mouse brains infected with the parental and the cDNA-derived viruses revealed a similar pathogenesis degree. We observed meningoencephalitis, cellular pyknosis, and neutrophilic invasion adjacent to the choroid plexus and perivascular cuffs with the presence of neutrophils. The developed infectious clone will be a tool for genetic and functional studies in vitro and in vivo to understand viral infection and pathogenesis better.

Differential Expression of Viral Transcripts From Single-Cell RNA Sequencing of Moderate and Severe COVID-19 Patients and Its Implications for Case Severity.

With steady increase of new COVID-19 cases around the world, especially in the United States, health care resources in areas with the disease outbreak are quickly exhausted by overwhelming numbers of COVID-19 patients. Therefore, strategies that can effectively and quickly predict the disease progression and stratify patients for appropriate health care arrangements are urgently needed. We explored the features and evolutionary difference of viral gene expression in the SARS-CoV-2 infected cells from the bronchoalveolar lavage fluids of patients with moderate and severe COVID-19 using both single cell and bulk tissue transcriptome data. We found SARS-CoV-2 sequences were detectable in 8 types of immune related cells, including macrophages, T cells, and NK cells. We first reported that the SARS-CoV-2 ORF10 gene was differentially expressed in the severe vs. moderate samples. Specifically, ORF10 was abundantly expressed in infected cells of severe cases, while it was barely detectable in the infected cells of moderate cases. Consequently, the expression ratio of ORF10 to nucleocapsid (N) was significantly higher in severe than moderate cases (p = 0.0062). Moreover, we found transcription regulatory sequences (TRSs) of the viral leader sequence-independent fusions with a 5' joint point at position 1073 of SARS-CoV-2 genome were detected mainly in the patients with death outcome, suggesting its potential indication of clinical outcome. Finally, we identified the motifs in TRS of the viral leader sequence-dependent fusion events of SARS-CoV-2 and compared with that in SARS-CoV, suggesting its evolutionary trajectory. These results implicated potential roles and predictive features of viral transcripts in the pathogenesis of COVID-19 moderate and severe patients. Such features and evolutionary patterns require more data to validate in future.