Loss of RAS Mutations in Liquid Biopsies of Patients With Multi-Treated Metastatic Colorectal Cancer.

BACKGROUND: Liquid biopsy (LB) is a non-invasive tool to evaluate the heterogeneity of tumors. Since RAS mutations (RAS-mut) play a major role in resistance to antiepidermal growth factor receptor inhibitors (EGFR) monoclonal antibodies (Mabs), serial monitoring of RAS-mut with LB may be useful to guide treatment. The main aim of this study was to evaluate the prognostic value of the loss of RAS-mut (NeoRAS-wt) in LB, during the treatment of metastatic colorectal cancer (mCRC). METHODS: A retrospective study was conducted on patients with mCRC between January 2018 and December 2021. RAS-mut were examined in tissue biopsy, at mCRC diagnosis, and with LB, during treatment. RESULTS: Thirty-nine patients with RAS-mut mCRC were studied. LB was performed after a median of 3 lines (0-7) of systemic treatment including anti-vascular endothelial growth factor (anti-VEGF) Mabs. NeoRAS-wt was detected in 13 patients (33.3%); 9 (69.2%) of them received further treatment with anti-EGFR Mabs with a disease control rate of 44.4%. Median overall survival (OS), from the date of LB testing, was 20 months in the NeoRAS-wt group and 9 months in the persistent RAS-mut group (log-rank 2.985; P = .08), with a 12-month OS of 84.6% and 57.7%, respectively. NeoRAS-wt was identified as a predictor of survival (HR = 0.29; P = .007), with an 11-month improvement in median OS and a 71% decrease in risk of death, in heavily pretreated patients. CONCLUSIONS: In conclusion, monitoring clonal evolution in mCRC by LB may provide an additional treatment line for patients with NeoRAS-wt in advanced disease.

Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage-defining mutations.

The ARTIC protocol uses a multiplexed PCR approach with two primer pools tiling the entire SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genome. Primer pool updates are necessary for accurate amplicon sequencing of evolving SARS-CoV-2 variants with novel mutations. The suitability of the ARTIC V4 and updated V4.1 primer scheme was assessed using whole genome sequencing of Omicron from clinical samples using Oxford Nanopore Technology. Analysis of Omicron BA.1 genomes revealed that 93.22 % of clinical samples generated improved genome coverage at 50× read depth with V4.1 primers when compared to V4 primers. Additionally, the V4.1 primers improved coverage of BA.1 across amplicons 76 and 88, which resulted in the detection of the variant-defining mutations G22898A, A26530G and C26577G. The Omicron BA.2 sub-variant (VUI-22JAN-01) replaced BA.1 as the dominant variant by March 2022, and analysis of 168 clinical samples showed reduced coverage across amplicons 15 and 75. Upon further interrogation of primer binding sites, a mutation at C4321T [present in 163/168 (97 %) of samples] was identified as a possible cause of complete dropout of amplicon 15. Furthermore, two mutations were identified within the primer binding regions for amplicon 75: A22786C (present in 90 % of samples) and C22792T (present in 12.5 % of samples). Together, these mutations may result in reduced coverage of amplicon 75, and further primer updates would allow the identification of the two BA.2-defining mutations present in amplicon 75: A22688G and T22679C. This work highlights the need for ongoing surveillance of primer matches as circulating variants evolve and change.

Lymph node yield in the pathological staging of resected nonmetastatic colon cancer: The more the better?

INTRODUCTION: Guidelines recommend regional lymphadenectomy with a lymph node yield (LNY) of at least 12 lymph nodes (LN) for adequate colon cancer (CC) staging. LNY ≥22LN may improve survival, especially in right-sided CC [Lee et al., Surg Oncol, 27(3), 2018]. This multicentric retrospective cohort study evaluated the impact of LNY and tumor laterality on CC staging and survival. MATERIALS AND METHODS: Patients with stage I-III CC that underwent surgery from 2012 to 2018 were grouped according to LNY: <22 and ≥ 22. Primary outcomes were LN positivity (N+ rate) and disease-free survival (DFS). Overall survival (OS) was the secondary outcome. Exploratory analyses were performed for laterality and stage. RESULTS: We included 795 patients (417 < 22LN, 378 ≥ 22LN); 53% had left-sided CC and 29%/37%/38% had stage I/II/III tumors. There was no association between LNY ≥22LN and N+ rate after adjustment for grade, T stage, lymphovascular invasion (LVI) and perineural invasion; a trend for a higher N+ rate in left-sided CC was identified (interaction p = 0.033). With a median follow-up of 63.6 months for DFS and 73.2 months for OS, 254 patients (31.9%) relapsed and 207 (26.0%) died. In multivariate analysis adjusted for age, ASA score, laparoscopic approach, T/N stage, mucinous histology, LVI and adjuvant chemotherapy, LNY ≥22LN was significantly associated with both DFS (HR 0.75, p = 0.031) and OS (HR 0.71, p = 0.025). Restricted cubic spline analysis showed a more significant benefit for right-sided CC. CONCLUSION: LNY ≥22LN was associated with longer DFS and OS in patients with operable CC, especially for right-sided CC.

Synthetic reconstruction of the hunchback promoter specifies the role of Bicoid, Zelda and Hunchback in the dynamics of its transcription.

For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as a model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid's partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.

Invasive Pulmonary Aspergillosis after COVID-19 Pneumonia.

COVID-19 emerged in China in late 2019 and quickly spread worldwide. The severe immunomodulation and depletion of lymphocytes caused by the virus and its therapy led to an increase in the incidence of superinfections. COVID-19-associated pulmonary aspergillosis (CAPA) is a new entity with increasing incidence and high associated mortality. We present the case of a 68-year-old patient admitted to our ward after recovering from severe COVID-19 pneumonia. Due to worsening of her clinical condition, chest computed tomography was performed and a lung abscess was documented with the identification of Aspergillus niger. Despite therapy with voriconazole, the patient's condition deteriorated, culminating in her death. LEARNING POINTS: COVID-19-associated pulmonary aspergillosis (CAPA) is a new entity with an increasing incidence.It is a serious and life-threatening complication in patients with severe COVID-19 even in the absence of the classic risk factors for invasive pulmonary aspergillosis.Clinical suspicion is crucial since a timely diagnosis and treatment have a major impact on prognosis.

SARS-CoV-2 Antibody Seroprevalence in Patients With Cancer on Systemic Antineoplastic Treatment in the First Wave of the COVID-19 Pandemic in Portugal.

At the time of the first wave of the COVID-19 pandemic, patients with cancer were considered to be at high risk of serious illness and had a higher exposure risk since they needed frequent and nondeferrable hospital visits. Serological tests were not routinely used, and seroprevalence in this population was unknown. A single-center, cross-sectional study was developed to determine the seroprevalence of anti-SARS-CoV-2 antibodies (Abs) in patients with cancer undergoing systemic antineoplastic treatment. One hundred patients were consecutively recruited in a two-week period (6th-20th May 2020), and serum samples were tested for the presence of immunoglobulin M (IgM) and immunoglobulin G (IgG) Abs directed against both spike (S) and nucleocapsid (N) SARS-CoV-2 proteins in two distinct time points (at recruitment and 4-8 weeks later). IgG-positive results were subject to confirmation, in the same serum sample, using two distinct assays. At the time of the first study visit, no patient had a previously confirmed diagnosis of COVID-19, one reported previous contact with a COVID-19 patient, and all had a baseline SARS-CoV-2-negative RT-PCR. Two patients tested positive for SARS-CoV-2 IgG in the first study visit, which was not confirmed in either of the two confirmatory assays. Seventy-two patients were tested at the second study visit, all with negative IgG tests. IgM was persistently positive at both study visits in one patient and was positive in another patient at the second study visit, both with negative RT-PCR and serum IgG. No patient tested positive for RT-PCR within the study timeframe. No evidence of prior or acute SARS-CoV-2 infection was documented in this cohort of patients with cancer undergoing systemic treatment, and no additional exposure risk was documented compared to general population seroprevalence studies. The study was inconclusive regarding the role of SARS-CoV-2 serology in patients with cancer in the early phase of the pandemic. This study did show that, with adherence to recommended preventive measures, it was safe to maintain systemic cancer therapy.

Switch to EGFR-TKI after upfront platinum doublet induction therapy in non-small cell lung cancer (NSCLC) patients with EGFR (Epidermal Growth Factor Receptor) mutation: A multicentre retrospective study.

Introduction of EGFR-TKI has changed the treatment paradigm for NSCLC patient with activating mutations of EGFR exons 18-21, replacing chemotherapy as standard first line treatment. Given the delays in molecular study results we sometimes face the need to start treatment in very symptomatic patients with high tumor burden. The reason for this retrospective study is to analyze the survival impact of performing an induction cytotoxic therapy until obtaining the molecular profile (EGFR mutation), followed by targeted therapy. This is a retrospective analysis of 31 patients who did upfront chemotherapy (ChT) before switching to EGFR TKI upon the molecular profile result. The calculated survival endpoints were progression-free survival (PFS), duration of TKI response and overall survival (OS). All patients were treated with upfront chemotherapy with a median of one cycle (range 1-3) followed by a first generation EGFR-TKI. Median PFS was 13 months (95% CI, 6.6-19.4) and median OS 33 months (95% CI, 11.9-54.0). After first line progression 14 patients were treated with Osimertinib. In this subgroup median OS was 52 months (95% CI, 34.0-69.9). In the multivariable Cox model, only body mass index retained independent prognostic significance for progression-free survival (p = 0.045). Survival outcomes in this cohort are in line with published data regarding first generation EGFR-TKI, both in terms of PFS and OS. Despite the limitations of this study, starting with upfront chemotherapy doesn't seem detrimental in terms of survival outcomes, with the potential advantage of symptomatic control. To our knowledge, this is the first study to address this strategy, which requires further confirmation.

Portuguese Football Federation consensus statement 2020: nutrition and performance in football.

Nutrition is an undeniable part of promoting health and performance among football (soccer) players. Nevertheless, nutritional strategies adopted in elite football can vary significantly depending on culture, habit and practical constraints and might not always be supported by scientific evidence. Therefore, a group of 28 Portuguese experts on sports nutrition, sports science and sports medicine sought to discuss current practices in the elite football landscape and review the existing evidence on nutritional strategies to be applied when supporting football players. Starting from understanding football's physical and physiological demands, five different moments were identified: preparing to play, match-day, recovery after matches, between matches and during injury or rehabilitation periods. When applicable, specificities of nutritional support to young athletes and female players were also addressed. The result is a set of practical recommendations that gathered consensus among involved experts, highlighting carbohydrates periodisation, hydration and conscious use of dietary supplements.

Falha na ativação da equipe de emergência intra-hospitalar: causas e consequências / Failure to activate the in-hospital emergency team: causes and outcomes

RESUMO Objetivo: Determinar a incidência de falha na ativação da via aferente da Equipe de Emergência Médica intra-hospitalar, caraterizando-a e comparando a mortalidade dessa população com a da população em que não se verificou falha na ativação da via aferente. Métodos: Entre janeiro de 2013 e julho de 2015, ocorreram 478 ativações da Equipe de Emergência Médica do Hospital Pedro Hispano. Após a exclusão de registos incompletos e ativações para doentes com menos de 6 horas de internamento hospitalar, obtivemos uma amostra de 285 ativações. A amostra foi dividida em dois grupos: o grupo com falha na ativação da via aferente e o grupo em que não ocorreu falha na ativação da via aferente da Equipe de Emergência Médica. As duas populações foram caracterizadas e comparadas. A significância estatística foi considerada para p ≤ 0,05. Resultado: Em 22,1% das ativações, verificou-se falha na ativação da via aferente. Relativamente ao estudo causal, verificamos existir diferença estatisticamente significativa quanto aos critérios de ativação da Equipe de Emergência Médica (p = 0,003) no grupo com falha na ativação da via aferente, encontrando taxa mais elevada de ativação da Equipe de Emergência Médica por paragem cardiorrespiratória e disfunção cardiovascular. Em relação às consequências, no grupo em que ocorreu falha na ativação da via aferente houve uma maior taxa de mortalidade imediata e à data de alta hospitalar, sem significado estatístico. Não encontramos diferenças significativas com relação aos outros parâmetros. Conclusão: Nos doentes em que houve falha da ativação da via aferente da Equipe de Emergência Médica, a incidência de paragem cardiorrespiratória e a taxa de mortalidade foram maiores. Este estudo reforça a necessidade de as unidades de saúde investirem na formação de todos os profissionais de saúde sobre os critérios de ativação da Equipe de Emergência Médica e o funcionamento do sistema de resposta a emergência médica.

ABSTRACT Objective: To determine the incidence of afferent limb failure of the in-hospital Medical Emergency Team, characterizing it and comparing the mortality between the population experiencing afferent limb failure and the population not experiencing afferent limb failure. Methods: A total of 478 activations of the Medical Emergency Team of Hospital Pedro Hispano occurred from January 2013 to July 2015. A sample of 285 activations was obtained after excluding incomplete records and activations for patients with less than 6 hours of hospitalization. The sample was divided into two groups: the group experiencing afferent limb failure and the group not experiencing afferent limb failure of the Medical Emergency Team. Both populations were characterized and compared. Statistical significance was set at p ≤ 0.05. Result: Afferent limb failure was observed in 22.1% of activations. The causal analysis revealed significant differences in Medical Emergency Team activation criteria (p = 0.003) in the group experiencing afferent limb failure, with higher rates of Medical Emergency Team activation for cardiac arrest and cardiovascular dysfunction. Regarding patient outcomes, the group experiencing afferent limb failure had higher immediate mortality rates and higher mortality rates at hospital discharge, with no significant differences. No significant differences were found for the other parameters. Conclusion: The incidence of cardiac arrest and the mortality rate were higher in patients experiencing failure of the afferent limb of the Medical Emergency Team. This study highlights the need for health units to invest in the training of all healthcare professionals regarding the Medical Emergency Team activation criteria and emergency medical response system operations.

A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels.

STUDY QUESTION: Could drugs targeting ATP-sensitive K(+) (K(ATP)) channels prevent any spontaneous increase in intracellular Ca(2+) that may occur in human metaphase II (MII) oocytes under in vitro conditions? SUMMARY ANSWER: Pinacidil, a K(ATP) channel opener, and glibenclamide, a K(ATP) channel blocker, prevent a spontaneous increase in intracellular Ca(2+) in human MII oocytes. WHAT IS KNOWN ALREADY: The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca(2+) homeostasis is crucial for cell wellbeing and increased intracellular Ca(2+) levels is a well-established indicator of cell stress. STUDY DESIGN, SIZE, DURATION: Supernumerary human oocytes (n = 102) collected during IVF/ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca(2+) levels were monitored in oocytes in the following experimental groups: (i) Control, (ii) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), (iii) Pinacidil, (iv) Glibenclamide, (v) DNP: an inhibitor of oxidative phosphorylation, (vi) Pinacidil and DNP and (vii) Glibenclamide and DNP. PARTICIPANTS/MATERIALS/SETTINGS/METHODS: Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean ± SD) age 34.1 ± 0.6 years, n = 41. Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca(2+)-sensitive dye, and monitored by laser confocal microscopy for 2 h at 10 min intervals. Time between oocyte collection and start of Ca(2+) monitoring was 80.4 ± 2.1 h. MAIN RESULTS AND THE ROLE OF CHANCE: Intracellular levels of Ca(2+) increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0 ± 11.8 AU (AU = arbitrary units; n = 23) to 91.8 ± 14.0 AU (n = 19; P < 0.001) after 2 h of monitoring. Pinacidil (100 µM) inhibited this increase in Ca(2+) (85.3 ± 12.3 AU at the beginning of the experiment, 81.7 ± 11.0 AU at the end of the experiment; n = 13; P = 0.616). Glibenclamide (100 µM) also inhibited the increase in Ca(2+) (74.7 ± 10.6 AU at the beginning and 71.8 ± 10.9 AU at the end of the experiment; n = 13; P = 0.851. DNP (100 mM) induced an increase in intracellular Ca(2+) that was inhibited by glibenclamide (100 µM; n = 9) but not by pinacidil (100 µM; n = 5). LIMITATIONS, REASONS FOR CAUTION: Owing to clinical and ethical considerations, it was not possible to monitor Ca(2+) in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4 ± 2.1 h (n = 102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilized, it is possible that they may have been abnormal with impaired Ca(2+) homeostasis and, furthermore, the altered Ca(2+) homeostasis might have been associated solely with the protracted incubation. WIDER IMPLICATIONS OF THE FINDINGS: These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca(2+), which can be counteracted by drugs targeting K(ATP) channels. As Ca(2+) homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that K(ATP) channel openers and blockers should be tested as drugs for improving success rates of ART. STUDY FUNDING/COMPETING INTERESTS: University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside. Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. The authors declare no conflicts of interest.

Failure to activate the in-hospital emergency team: causes and outcomes. / Falha na ativação da equipe de emergência intra-hospitalar: causas e consequências.

OBJECTIVE:: To determine the incidence of afferent limb failure of the in-hospital Medical Emergency Team, characterizing it and comparing the mortality between the population experiencing afferent limb failure and the population not experiencing afferent limb failure. METHODS:: A total of 478 activations of the Medical Emergency Team of Hospital Pedro Hispano occurred from January 2013 to July 2015. A sample of 285 activations was obtained after excluding incomplete records and activations for patients with less than 6 hours of hospitalization. The sample was divided into two groups: the group experiencing afferent limb failure and the group not experiencing afferent limb failure of the Medical Emergency Team. Both populations were characterized and compared. Statistical significance was set at p ≤ 0.05. RESULT:: Afferent limb failure was observed in 22.1% of activations. The causal analysis revealed significant differences in Medical Emergency Team activation criteria (p = 0.003) in the group experiencing afferent limb failure, with higher rates of Medical Emergency Team activation for cardiac arrest and cardiovascular dysfunction. Regarding patient outcomes, the group experiencing afferent limb failure had higher immediate mortality rates and higher mortality rates at hospital discharge, with no significant differences. No significant differences were found for the other parameters. CONCLUSION:: The incidence of cardiac arrest and the mortality rate were higher in patients experiencing failure of the afferent limb of the Medical Emergency Team. This study highlights the need for health units to invest in the training of all healthcare professionals regarding the Medical Emergency Team activation criteria and emergency medical response system operations.

[Abdominal compartment syndrome: survey on the awareness of Portuguese general surgeons]. / Síndrome de compartimento abdominal: questionário sobre a sensibilidade dos cirurgiões gerais Portugueses.

The Abdominal Compartment Syndrome (ACS) is a clinical entity recognized for over a century, but only recently its risk criteria, monitorization and treatment have been defined by the World Society of the Abdominal Compartment Syndrome (WSACS). The general surgeon's involvement is vital since this syndrome is common in surgical patients and because its treatment may culminate in a laparostomy. 250 questionnaires of 17 questions were distributed among general surgeons attending the XXVIII Portuguese Congress of Surgery. The data were analyzed using SPSS® v16. We received 36,4% (91) of the delivered questionnaires, most of which from male surgeons (63,7%), from central hospitals (75,8%), working 42 h per week (70.3%), whose average of age was 38 years. About half of the respondents received training in Intensive Care Units. All surgeons had already heard about measuring the Intra- abdominal Pressure (IAP), which was being performed at 89% of their hospitals. About 40% of surgeons only admitted intra-abdominal hypertension above 20 mmHg (only 22% indicated the correct value of 12 mmHg). 36,3% of surgeons suggested that a decompressive laparostomy must be carried out for primary ACS if IAP greater then 20 mmHg with new organ failure; 36.3% favoured the "Vacuum-pack"-like system, and 56% only re-operate the patients "as needed". 48,4% of surgeons had already performed decompressive laparostomy, 66% of which had residence training in a ICU (p = 0,005). Respondents also pointed an average mortality related to ACS of 81% without laparostomy, and a reduction to 38,5% after performing that procedure. Only 26% of the surgeons were aware of the WSACS consensus definitions and recommendations, of those, 83% had already performed a laparostomy (P<0,001). It can be concluded that, in spite of recognizing the ACS as a clinical entity, portuguese general surgeons are quite unaware of the WSACS definitions and treatment guidelines, urging the need for its divulgation.