Mutational spectrum and deep phenotyping in Pseudoxanthoma Elasticum: Findings from a Portuguese cohort.

INTRODUCTION: Pseudoxanthoma Elasticum (PXE) is a rare autosomal recessive disorder originated by disease-causing variants in ABCC6 gene. The purpose of this study was to characterize the genetic landscape, phenotypic spectrum and genotype-phenotype correlations in a Portuguese cohort of PXE patients. METHODS: Multicentric cross-sectional study conducted in patients with a clinical and genetic diagnosis of PXE. Patients were identified using the IRD-PT registry (www.retina.com.pt). Genotypes were classified into 3 groups: (1) two truncating variants, (2) two non-truncating variants, or (3) mixed variants. Deep phenotyping comprised a comprehensive ophthalmologic and systemic evaluation using the updated Phenodex Score (PS). RESULTS: Twenty-seven patients (23 families) were included. Sixteen different ABCC6 variants were identified, 7 of which are novel. The most prevalent variant was the nonsense variant c.3421C > T p.(Arg1141*) with an allele frequency of 18.5%. All patients exhibited ocular manifestations. Cutaneous manifestations were present in most patients (88.9%, n = 24/27). A PS score > E2 was strongly associated with worse visual acuity (B = -29.02; p = 0.001). No association was found between genotypic groups and cutaneous, vascular or cardiac manifestations. CONCLUSIONS: This study describes the genetic spectrum of patients with PXE for the first time in a Portuguese cohort. A total of 16 different variants in ABCC6 were found (7 of which are novel), thus highlighting the genotypic heterogeneity associated with this condition and expanding its mutational spectrum. Still, no major genotype-phenotype associations could be established.

Characteristics and patient-reported outcomes of long-term lung cancer survivors.

Background: Due to advances in screening and treatment of lung cancer, there has been increased interest in long-term lung cancer survivors (LTLCS). The aim of this study was to evaluate the prevalence of LTLCS, their characteristics and patient-reported outcomes (PROs) of LTLCS. Methods: Cross-sectional study that included patients diagnosed with primary lung cancer between Jan 2012 and Dec 2016 whose overall survival (OS) was greater than 5 years. A self-administered questionnaire was applied, including European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core-30 (EORTC QLQ-C30), Patient Health Questionnaire-4 (PHQ-4) and two open questions regarding quality of life (QoL) and suggestions for improvements. Factors potentially related to QoL were analysed. Results: Of 767 lung cancer patients, 158 (20.6%) were LTLCS and LTLCS' proportion increased yearly. Most patients were male (70.9%) with median age of 65 [interquartile range (IQR), 56-71] years. Fifty-seven percent had adenocarcinoma, 66.2% were diagnosed at early stages but 8.9% were at stage IV. During follow-up, 77.1% quitted smoking, 31.8% had disease progression/relapse and 15.2% developed other tumours. Of all living LTLCS, 100 (85%) patients answered the PROs questionnaire. The median Global Health score was 66.67 (IQR, 50-83), social functioning had the best score and emotional functioning the worst. Pain and fatigue were the symptoms with the worst impact on QoL. PHQ-4 identified mental distress in 36% and patients with a lower QoL were more likely to present anxiety (35.3% vs. 9.4%, P=0.007) or depression (27.9% vs. 3%, P=0.006). In the open questions, patients reported pain (17%), lack of familiar/financial support (16%), dyspnoea (14%), depression (8%), concern for the future (8%) and limitations performing daily activities (8%) as the aspects with most impact in QoL. The most suggested measures were improvement of care provided by health institutions (25%) and better social support (16%). Conclusions: Prevalence of LTLCS is increasing and survivors may experience a high prevalence of anxiety and depression as well as a high disease burden affecting QoL. Therefore, it's important to provide multidisciplinary continuous patient-centred care and a careful follow-up for all lung cancer patients, including LTLCS.

ERS International Congress 2023: highlights from the Thoracic Oncology Assembly.

Lung cancer is the leading cause of cancer mortality in the world. It greatly affects the patients' quality of life, and is thus a challenge for the daily practice in respiratory medicine. Advances in the genetic knowledge of thoracic tumours' mutational landscape, and the development of targeted therapies and immune checkpoint inhibitors, have led to a paradigm shift in the treatment of lung cancer and pleural mesothelioma. During the 2023 European Respiratory Society Congress in Milan, Italy, experts from all over the world presented their high-quality research and reviewed best clinical practices. Lung cancer screening, management of early stages of lung cancer, application of artificial intelligence and biomarkers were discussed and they will be summarised here.

Mental health conditions and utilisation of mental health services by survivors of physical intimate partner violence in Portugal: Results from the WHO world mental health survey.

The aim of this article is to study mental health conditions among survivors of severe physical intimate partner violence (IPV) and their utilisation of mental health services. This study is an integrated part of the World Mental Health Survey Initiative-Portugal, for which data was collected from a nationally representative adult sample using well-validated scales. Logistic regression models were used in the analysis. The most common statistically significant mental health conditions among IPV survivors were suicide ideation, PTSD, major depressive episode, and generalised anxiety disorder. More than one in three survivors developed PTSD. Suicide ideation was likely to occur after first experiencing IPV. Almost a half of survivors received specialised mental health treatment; in most cases, delivered by a psychiatrist. Over 60 % addressed their mental health issues consulting general physicians or other healthcare professionals. Those who experienced family violence in childhood had greater odds of also experiencing IPV; survivors of IPV with this experience were more likely to receive mental health treatment. The need to promote greater awareness and competencies of not only mental health professionals but also of general physicians and other healthcare professionals to provide support more effectively to survivors of any type of IPV deserves to be emphasised.

Current Applications and Challenges of Next-Generation Sequencing in Plasma Circulating Tumour DNA of Ovarian Cancer.

Circulating tumour DNA (ctDNA) facilitates longitudinal study of the tumour genome, which, unlike tumour tissue biopsies, globally reflects intratumor and intermetastatis heterogeneity. Despite its costs, next-generation sequencing (NGS) has revolutionised the study of ctDNA, ensuring a more comprehensive and multimodal approach, increasing data collection, and introducing new variables that can be correlated with clinical outcomes. Current NGS strategies can comprise a tumour-informed set of genes or the entire genome and detect a tumour fraction as low as 10-5. Despite some conflicting studies, there is evidence that ctDNA levels can predict the worse outcomes of ovarian cancer (OC) in both early and advanced disease. Changes in those levels can also be informative regarding treatment efficacy and tumour recurrence, capable of outperforming CA-125, currently the only universally utilised plasma biomarker in high-grade serous OC (HGSOC). Qualitative evaluation of sequencing shows that increasing copy number alterations and gene variants during treatment may correlate with a worse prognosis in HGSOC. However, following tumour clonality and emerging variants during treatment poses a more unique opportunity to define treatment response, select patients based on their emerging resistance mechanisms, like BRCA secondary mutations, and discover potential targetable variants. Sequencing of tumour biopsies and ctDNA is not always concordant, likely as a result of clonal heterogeneity, which is better captured in the plasma samples than it is in a large number of biopsies. These incoherences may reflect tumour clonality and reveal the acquired alterations that cause treatment resistance. Cell-free DNA methylation profiles can be used to distinguish OC from healthy individuals, and NGS methylation panels have been shown to have excellent diagnostic capabilities. Also, methylation signatures showed promise in explaining treatment responses, including BRCA dysfunction. ctDNA is evolving as a promising new biomarker to track tumour evolution and clonality through the treatment of early and advanced ovarian cancer, with potential applicability in prognostic prediction and treatment selection. While its role in HGSOC paves the way to clinical applicability, its potential interest in other histological subtypes of OC remains unknown.

Prevalence and acceptability of psychological and/or economic intimate partner violence, and utilization of mental health services by its survivors in Lithuania.

BACKGROUND: Lithuania has one of the highest averages in the European Union when it comes to psychological and/or economic intimate partner violence (PE-IPV). IPV survivors are several times more likely to have mental health conditions than those without IPV experiences. The aim of this article is to study the prevalence, characteristics and attitudes of PE-IPV survivors in Lithuania, and the predictors of them accessing mental health services. METHODS: A cross-sectional study based on a national survey representative of the adult population. The survey was implemented by a third-party independent market research company employing an online survey panel. Logistic regression models were used in the analysis. RESULTS: Almost 50% of women in Lithuania experience PE-IPV. Females are significantly more likely to experience it than males. The vast majority of women find PE-IPV unacceptable; however, only one-third of survivors seek any type of help. Only one-tenth approach mental health services, with divorcees being at higher odds of doing so. CONCLUSIONS: Further research is needed to explore predictors and contextual factors of why IPV survivors seek mental healthcare, or not. Policy implications include the need to eliminate IPV and mental health stigma; develop accessible mental health services and effective treatment approaches.

The Concept behind the Suitability of Menstrual Blood-Derived Stem Cells for the Management of Vaginal Atrophy among BRCA Mutation Carriers after RRSO.

Risk-reducing bilateral salpingo-oophorectomy (RRSO) is recommended for breast cancer gene 1 (BRCA1) and 2 (BRCA2) mutation carriers. A major consequence of RRSO is surgical menopause associated with severe menopausal symptoms, mostly genitourinary complaints. Due to the inherent breast cancer risk, estrogen-based therapies are generally avoided in these patients. So far, the non-hormonal approaches available are not efficient to successfully treat the disabling vaginal atrophy-related symptoms. In regenerative medicine, mesenchymal stem cells (MSC) are the most frequently used cell type due to their remarkable and regenerative characteristics. Therapies based on MSC have revealed positive outcomes regarding symptoms and signs associated with vaginal atrophy by promoting angiogenesis, vaginal restoration, and the proliferation of vaginal mucosa cells. Menstrual blood-derived stem cells (MenSC) are a novel source of MSC, with promising therapeutic potential directly linked to their high proliferative rates; low immunogenicity; non-invasive, easy, and periodic acquisition; and almost no associated ethical issues. In this review, we update the current knowledge and research regarding the potential value of previously preserved MenSC in the therapy of vaginal atrophy among BRCA mutation carriers subjected to RRSO.

Use of coercion in mental healthcare services in Nigeria: Service providers' perspective.

BACKGROUND: There is increasing advocacy to reduce coercive practices in mental healthcare. Little research has been done on the topic in developing countries. AIMS: To explore what mental health professionals in Nigeria think about coercion, why it is used, and what contextual factors influence its use in mental healthcare services. METHODS: Semi-structured interviews were carried out with 16 doctors and 14 nurses from two psychiatric hospitals in two regions of Nigeria. The audio-recorded interviews were analyzed thematically with the aid of MAXQDA software. RESULTS: Three broad categories relating to the perception of, reasons for, and barriers/facilitators to the use of coercion were derived. Coercion was viewed as being for the best interests of patients, a means to an end, and effective for achieving desired outcomes. Safety was both a reason to use a coercive measure and a deterrent to using specific coercive measures thought to be unsafe. The socio-cultural context, obsolete mental health legislation, staff shortages, and attitudes were factors influencing the use of coercion in mental healthcare. CONCLUSION: Coercion was perceived to be ultimately beneficial in mental health practice. There is need for a new mental health legislation and more resources for mental health care in Nigeria to address the problem of coercion.

Endometrial fluid biomarkers and their potential as predictors of successful embryo implantation.

Background: Embryo implantation is a complex biological process which requires synchronized dialogue between the receptive endometrium and the blastocyst. The endometrium, however, is only receptive to embryo implantation for a very short period. Recurrent implantation failure (RIF) is a major challenge in assisted reproductive techniques mainly due to impaired receptivity, but there is still a need for a reliable and valid clinical test to assess endometrial receptiveness, especially at embryo transfer time. The aim of this review is to investigate what is currently known about the contribution of endometrial fluid (EF) to endometrial receptivity by identifying its potential biomarkers. Methods: This study involved an extensive search of the electronic databases PubMed and Cochrane, covering the period from 2011 to 2022. A combination of Medical Subject Headings with the terms 'endometrial fluid' and 'embryo implantation' was used. Results: Several different proteins presented in the endometrial cavity fluid have been described but the most consistent as potential biomarkers were Proprotein Convertase 6 (PC6), Vascular Endothelial Growth Factor (VEGF), PIGF (Placental growth factor), ß3 integrin, Colony Stimulating Factor-3 (CSF-3), Leukaemia inhibitory factor (LIF), glycodelin and extracellular vesicles (EVs). Conclusions: Strong indicators support the use of uterine fluid collection as a non-invasive tool for receptivity assessment. Therefore, it could improve outcomes of assisted reproductive techniques.

New Insights into Photobiomodulation of the Vaginal Microbiome-A Critical Review.

The development of new technologies such as sequencing has greatly enhanced our understanding of the human microbiome. The interactions between the human microbiome and the development of several diseases have been the subject of recent research. In-depth knowledge about the vaginal microbiome (VMB) has shown that dysbiosis is closely related to the development of gynecologic and obstetric disorders. To date, the progress in treating or modulating the VMB has lagged far behind research efforts. Photobiomodulation (PBM) uses low levels of light, usually red or near-infrared, to treat a diversity of conditions. Several studies have demonstrated that PBM can control the microbiome and improve the activity of the immune system. In recent years, increasing attention has been paid to the microbiome, mostly to the gut microbiome and its connections with many diseases, such as metabolic disorders, obesity, cardiovascular disorders, autoimmunity, and neurological disorders. The applicability of PBM therapeutics to treat gut dysbiosis has been studied, with promising results. The possible cellular and molecular effects of PBM on the vaginal microbiome constitute a theoretical and promising field that is starting to take its first steps. In this review, we will discuss the potential mechanisms and effects of photobiomodulation in the VMB.

Contextual factors influencing the use of coercive measures in Portuguese mental health care.

The use of coercive measures in mental health care is an important indicator of the quality of care being provided, and non-patient-related factors are increasingly recognized to contribute to their use. The study aimed to explore the perspectives of mental health care professionals who have first-hand experience with the use of coercion on the contextual factors that influence the use of coercion in the Portuguese mental health care. Five focus group discussions were conducted among 23 doctors and 17 nurses from five psychiatric departments in urban and rural regions of Portugal. Discussions were audio recorded, transcribed, and analyzed with the aid of MAXQDA. Four broad themes related to insufficient resources, staff-related factors, inefficient services, and socio-legal factors were derived. Participants highlighted how inadequate structures, staff shortages, staff attitudes, a lack of training, restrictive ward rules, an inefficient organization of services, the mental health legislation, and public attitudes contributed to the use of coercive measures. The COVID-19 pandemic complicated existing shortfalls in the system and increased the use of coercive measures. The study confirms that the use of coercive measures in mental health care is influenced by factors that are independent of patient characteristics. Addressing existing systemic problems is crucial for the successful implementation of interventions to reduce coercion in mental health care.

Seminal human papillomavirus infection: a narrative review.

BACKGROUND: Human papillomavirus (HPV) infection is one of the most prevalent sexually transmitted infections that can lead to malignant pathologies as well as fertility problems. The aim of this study was to evaluate the prevalence of HPV infection in males, its impact on semen parameters, and reproductive consequences. We also evaluated potential measures that could prevent negative outcomes of HPV infection in males. METHODS: A systematic literature search using PubMed/Medline and Embase databases was performed to search for English articles published until July 2023. We explored three different aspects: (1) prevalence of HPV semen infection and its impact on seminal parameters; (2) the relationship between HPV semen infection and infertility risk and reproductive outcomes; and (3) potential measures that could prevent the negative outcomes related to HPV seminal infection. The identified studies were first screened and assessed independently by one author, and then validated by two additional authors. Data were extracted from 19 studies. RESULTS: The prevalence of seminal HPV infection was higher among infertile males. In addition to controversies about the real interference of seminal HPV infection on sperm parameters, a growing number of studies have demonstrated a correlation between unexplained infertility and seminal HPV infection. Semen HPV infection is also associated with lower rates of pregnancy and higher rates of miscarriage. Prevention measures such as HPV vaccination seem promising. CONCLUSION: Further studies are required to confirm not only the association between HPV infection and reproductive outcomes but also the benefit of preventive measures.

Orofacial clefts: Reflections on prenatal diagnosis and family history based on a series of cases of a tertiary children hospital.

Prenatal diagnosis of orofacial clefts allows adequate counseling and planning for prenatal care and delivery. In 2001, two-dimensional ultrasound screening became universally used in Portugal by government guidelines, and after 2007 more advanced ultrasound became available. This study aimed to describe the prevalence of family history in patients with orofacial clefts and analyze prenatal diagnosis in patients born before 2001, between 2001 and 2007 and after 2007. Retrospective analysis of a cohort of patients with orofacial clefts followed by the trans-disciplinary team of a tertiary hospital. A total of 672 OFCs were identified: 40.9% isolated cleft palate, 38.1% cleft lip and palate, 19.7% cleft lip and 1.3% atypical cleft; 57.1% were male. The prevalence of family history was 26.0% of which 30.9% had a recognizable syndrome. Of those born before 2001, 13.7% had prenatal diagnosis; of those born between 2001 and 2007, 32.6% orofacial clefts were diagnosed in utero; and in children born after 2007, prenatal diagnosis increased to 47.1%. In our study, about one-fourth of children had a positive family history. Since the implementation of universal ultrasound screening in Portugal, more orofacial clefts were identified in utero (42.5% vs. 13.7%; p < 0.05) and after the availability of advanced ultrasound, prenatal diagnosis increased to 47.1% (vs. 20.4% before 2007; p < 0.05). Of all orofacial clefts diagnosed prenatally, ultrasound revealed more accuracy for the diagnosis of cleft lip and palate (65.4%) and cleft lip (24.8%). Cleft palate is the most difficult to detect in utero (9.3%). Prenatal ultrasound screening in Portugal has technically evolved with consequent better diagnostic accuracy for the identification of orofacial clefts, allowing better parenteral counseling.

Beyond Patient Characteristics: A Narrative Review of Contextual Factors Influencing Involuntary Admissions in Mental Health Care.

Variations in the rates of involuntary admission (IA) reflect the influence of unexplained contextual variables that are typically too heterogeneous to be included in systematic reviews. This paper attempts to gather and analyze factors unrelated to the patients that have been linked to IA. The articles included in this review were selected by iteratively searching four electronic databases (PubMed, PsychINFO, EMBASE, and Web of Science). A total of 54 studies from 19 different countries and regions, including 14 European countries, the United States, Canada, China, Vietnam, and Taiwan, were selected. The factors were categorized as service-related factors, impactful events, seasonal and temporal factors, mental health legislation, staff factors, and public attitudes. The factors rarely act in isolation but rather interact and reinforce each other, causing a greater influence on IA. This paper explains how these factors present opportunities for robust and sustainable interventions to reduce IAs. The paper also identifies future directions for research, such as examining the effects of economic recessions. Enhancing global reporting standards is essential to validate future research and support further in-depth studies. The complexity of the factors influencing IA and the implicit role of society suggest that resolving it will require social change.

Marking Techniques for Targeted Axillary Dissection Among Patients With Node-Positive Breast Cancer Treated With Neoadjuvant Chemotherapy.

Invasive breast cancer with axillary lymph node (LN) invasion is a continuing problem worldwide. The morbidity associated with axillary LN dissection along with the high rate of nodal downstaging after neoadjuvant chemotherapy (NACT) made the standard treatment shift towards less invasive surgery. Sentinel lymph node biopsy (SLNB) after NACT is associated with high false-negative rates (13%-14%). To overcome this problem, it was concluded that the positive nodes should first be indicated with image-detectable markers and then removed together with SLNB: targeted axillary dissection (TAD). This review aims to describe and evaluate the different marking techniques for TAD in patients with node-positive breast cancer treated with NACT, namely: clip placement and guidewire localization; clip placement and 125I-labelled radioactive seed localization; clip placement and skin mark; clip placement and intraoperative ultrasound; tattooing with a sterile black carbon suspension; magnetic seeds; radar and infrared light technology localization. Targeted axillary dissection techniques have shown false-negative rates below 9% and identification rates above 95%. The most studied technique is guidewire localization, as it is also the oldest one. However, according to data gathered from this review, some newer techniques have shown to be very promising due to their statistical results and management factors.

A qualitative integrative analysis of service users' and service providers' perspectives on ways to reduce coercion in mental health care.

PURPOSE: The movement to develop and implement non-coercive alternatives in the provision of mental health care is gaining momentum globally. To strengthen the basis of potential interventions that will be contextually relevant, and to complement the body of literature which is largely from high-income settings, the current study sought to explore the suggestions of service users and providers in Nigeria on how to reduce the use of coercive measures in mental health settings. METHODS: Semi-structured interviews with 30 mental health professionals and four focus group discussions among 30 service users from two psychiatric hospitals in Nigeria were conducted. The data were analyzed thematically with the aid of MAXQDA. RESULTS: The suggestions proposed by service users and mental health professionals were within the broad themes of communication, policies and legislation, and increased resources. Service users felt that improved communication, home consultations, non-legal advocates and clear rules and legislation would reduce the use of coercion, while service providers suggested increased public mental health literacy, better interpersonal relationships with patients, increased resources for mental health care, more research on the topic and regulation of coercive measures. CONCLUSION: Many of the suggestions from this study reinforce strategies already in place to decrease coercion in other settings. However, additional recommendations that are relevant to the study setting, such as enhancing public mental health literacy, mental health legislation reform and increasing access to mental health services, deserve further consideration.

The Psychosocial Impact of the Decision to Undergo Risk-Reducing Salpingo-Oophorectomy Surgery in BRCA Mutation Carriers and the Role of Physician-Patient Communication.

Risk-reducing bilateral salpingo-oophorectomy (RRSO) is an effective prophylactic surgery provided to premenopausal women carrying BRCA1 or BRCA2 mutations and presenting an increased risk of developing breast or ovarian cancer. This procedure is related to physiological, sexual, and psychosocial distress, which altogether increase uncertainty and complexity in the clinical decision-making process and post-surgery adaptation. Physician-patient communication (PPC) has been pointed out as a determinant factor in the decision-making to undergo RRSO, and the subsequent adjustment of women. However, studies examining the psychosocial impact of the decision-making process have been scarce and often lack clear theoretical frameworks. While the role of PPC in such processes has been highlighted in a few qualitative studies, there is a paucity of quantitative research addressing this question. Therefore, this narrative review, conducted using a multidisciplinary approach, was planned to: (1) present an updated medical background for RRSO; (2) analyze the psychosocial impact of the decision-making process within a theoretical framework of the Health Belief Model; and (3) discuss the role of PPC in such a decision-making process and in post-surgery. The collected research also enabled the recommendation of some additions to the existing clinical guidelines and the outlining of future research directions.

Experiences of care perceived by users of supported accommodations for people with serious mental disorders: Can they impact quality of life?

BACKGROUND: To understand if supported accommodations (SA) are promoting the recovery of people with serious mental disorders, quality of life (QoL) is an important outcome. This study aimed to analyse the association between QoL and experiences of care in general and to identify specific experiences of care that are associated with QoL in users of SA. METHODS: A random sample of users of 42 SA was interviewed to obtain standardized measures of QoL and personal experiences of care. The sample was also characterized according to sociodemographic and clinical aspects. Linear regressions models analysed the association between QoL and experiences of care, adjusting for potential confounders. Results include estimated regression coefficients, corresponding 95% confidence intervals and p-values. RESULTS: The number of users interviewed was 272. The median QoL was 4.9 (2.3-6.8) out of 7. Although 84.9% of users were satisfied with the care received, only 16.2% felt involved in their treatment. Feeling safe (ß = .73; 95% CI [0.22-1.24], p = .006) and having privacy (ß = .42; 95% CI [0.09-0.75], p = .014) influenced QoL. Involvement in care (ß = .44; 95% CI [0.13-0.74], p = .006), safety and privacy (ß = .72; 95% CI [0.44-0.99], p = 8.38e-07) and user-professional relationship (ß = .42; 95% CI [0.14-0.69], p = .003) were also associated with QoL. CONCLUSIONS: Feeling safe, having privacy, feeling involved in care and having good user-professional relationships influence the QoL of users. These findings have implications from the political and economic level to the organizational and individual levels.

One Step Nucleic Acid Amplification (OSNA) Lysate Samples Are Suitable to Establish a Transcriptional Metastatic Signature in Patients with Early Stage Hormone Receptors-Positive Breast Cancer.

The One Step Nucleic Acid Amplification (OSNA) is being adopted worldwide for sentinel lymph nodes (SLNs) staging in breast cancer (BC). As major disadvantage, OSNA precludes prognostic information based on structural evaluation of SLNs. Our aim is to identify biomarkers related to tumor-microenvironment interplay exploring gene expression data from the OSNA remaining lysate. This study included 32 patients with early stage hormone receptors-positive BC. Remaining OSNA lysates were prepared for targeted RNA-sequencing analysis. Identification of differentially expressed genes (DEGs) was performed by DESeq2 in R and data analysis in STATA. The results show that, in metastatic SLNs, several genes were upregulated: KRT7, VTCN1, CD44, GATA3, ALOX15B, RORC, NECTIN2, LRG1, CD276, FOXM1 and IGF1R. Hierarchical clustering analysis revealed three different clusters. The identified DEGs codify proteins mainly involved in cancer aggressiveness and with impact in immune response. The overexpression of the immune suppressive genes VTCN1 and CD276 may explain that no direct evidence of activation of immune response in metastatic SLNs was found. We show that OSNA results may be improved incorporating microenvironment-related biomarkers that may be useful in the future for prognosis stratification and immunotherapy selection. As OSNA assay is being implemented for SLNs staging in other cancers, this approach could also have a wider utility.