Interaction between NSCLC Cells, CD8+ T-Cells and Immune Checkpoint Inhibitors Potentiates Coagulation and Promotes Metabolic Remodeling-New Cues on CAT-VTE.

BACKGROUND: Cancer-associated thrombosis (CAT) and venous thromboembolism (VTE) are frequent cancer-related complications associated with high mortality; thus, this urges the identification of predictive markers. Immune checkpoint inhibitors (ICIs) used in cancer immunotherapy allow T-cell activation against cancer cells. Retrospective studies showed increased VTE following ICI administration in some patients. Non-small cell lung cancer (NSCLC) patients are at high risk of thrombosis and thus, the adoption of immunotherapy, as a first-line treatment, seems to be associated with coagulation-fibrinolysis derangement. METHODS: We pharmacologically modulated NSCLC cell lines in co-culture with CD8+ T-cells (TCD8+) and myeloid-derived suppressor cells (MDSCs), isolated from healthy blood donors. The effects of ICIs Nivolumab and Ipilimumab on NSCLC cell death were assessed by annexin V and propidium iodide (PI) flow cytometry analysis. The potential procoagulant properties were analyzed by in vitro clotting assays and enzyme-linked immunosorbent assays (ELISAs). The metabolic remodeling induced by the ICIs was explored by 1H nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Flow cytometry analysis showed that TCD8+ and ICIs increase cell death in H292 and PC-9 cells but not in A549 cells. Conditioned media from NSCLC cells exposed to TCD8+ and ICI induced in vitro platelet aggregation. In A549, Podoplanin (PDPN) levels increased with Nivolumab. In H292, ICIs increased PDPN levels in the absence of TCD8+. In PC-9, Ipilimumab decreased PDPN levels, this effect being rescued by TCD8+. MDSCs did not interfere with the effect of TCD8+ in the production of TF or PDPN in any NSCLC cell lines. The exometabolome showed a metabolic remodeling in NSCLC cells upon exposure to TCD8+ and ICIs. CONCLUSIONS: This study provides some insights into the interplay of immune cells, ICIs and cancer cells influencing the coagulation status. ICIs are important promoters of coagulation, benefiting from TCD8+ mediation. The exometabolome analysis highlighted the relevance of acetate, pyruvate, glycine, glutamine, valine, leucine and isoleucine as biomarkers. Further investigation is needed to validate this finding in a cohort of NSCLC patients.

BRD9 status is a major contributor for cysteine metabolic remodeling through MST and EAAT3 modulation in malignant melanoma.

Cutaneous melanoma (CM) is the most aggressive skin cancer, showing globally increasing incidence. Hereditary CM accounts for a significant percentage (5-15 %) of all CM cases. However, most familial cases remain without a known genetic cause. Even though, BRD9 has been associated to CM as a susceptibility gene. The molecular events following BRD9 mutagenesis are still not completely understood. In this study, we disclosed BRD9 as a key regulator in cysteine metabolism and associated altered BRD9 to increased cell proliferation, migration and invasiveness, as well as to altered melanin levels, inducing higher susceptibility to melanomagenesis. It is evident that BRD9 WT and mutated BRD9 (c.183G>C) have a different impact on cysteine metabolism, respectively by inhibiting and activating MPST expression in the metastatic A375 cell line. The effect of the mutated BRD9 variant was more evident in A375 cells than in the less invasive WM115 line. Our data point out novel molecular and metabolic mechanisms dependent on BRD9 status that potentially account for the increased risk of developing CM and enhancing CM aggressiveness. Moreover, our findings emphasize the role of cysteine metabolism remodeling in melanoma progression and open new queues to follow to explore the role of BRD9 as a melanoma susceptibility or cancer-related gene.

NSCLC presents metabolic heterogeneity, and there is still some leeway for EGF stimuli in EGFR-mutated NSCLC.

BACKGROUND: Metabolic remodeling is crucial in carcinogenesis and cancer progression. Oncogenic mutations may promote metabolic reprogramming in cancer cells to support their energy and biomass requirements. EGFR mutations are commonly found in non-small cell lung cancer (NSCLC) and may induce NSCLC metabolic rewiring. Whether EGFR-driven metabolic reprogramming triggers cell vulnerabilities with therapeutic potential remains unknown. METHODS: The role of EGFR signaling activation by EGF was investigated using NSCLC cell lines with different EGFR and KRAS status: A549 (EGFR WT and KRAS c.34G > A), H292 (EGFR WT and KRAS WT) and PC-9 (EGFR exon 19 E746-A750 deletion and KRAS WT). The effect of EGF on NSCLC cell death and cell cycle was evaluated using flow cytometry, and cell migration was assessed through wound healing. EGFR, HER2, MCT1, and MCT4 expression was analyzed through immunofluorescence or western blotting. We explored the impact of glucose and lactate bioavailability on NSCLC cells' metabolic profile using nuclear magnetic resonance (NMR) spectroscopy. Moreover, the expression of several relevant metabolic genes in NSCLC cells or patient samples was determined by RT-qPCR. RESULTS: We showed that cell lines presented different metabolic profiles, and PC-9 cells were the most responsive to EGF stimulus, as they showed higher rates of cell proliferation and migration, together with altered metabolic behavior. By inhibiting EGFR with gefitinib, a decrease in glucose consumption was observed, which may be related to the fact that despite PC-9 harbor EGFR mutation, they still express the EGFR WT allele. The analysis of NSCLC patients' RNA showed a correlation between MCT1/MCT4 and GLUT1 expression in most cases, indicating that the metabolic information can serve as a reference in patients' follow-up. CONCLUSION: Together, this study shows that NSCLC cell lines have heterogeneous metabolic profiles, which may be underlaid by different genetic profiles, revealing an opportunity to identify and stratify patients who can benefit from metabolism-targeted therapies.

H2S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine.

Lung cancer is a lethal disease with no truly efficient therapeutic management despite the progresses, and metabolic profiling can be a way of stratifying patients who may benefit from new therapies. The present study is dedicated to profiling cysteine metabolic pathways in NSCLC cell lines and tumor samples. This was carried out by analyzing hydrogen sulfide (H2S) and ATP levels, examining mRNA and protein expression patterns of cysteine catabolic enzymes and transporters, and conducting metabolomics analysis using nuclear magnetic resonance (NMR) spectroscopy. Selenium-chrysin (SeChry) was tested as a therapeutic alternative with the aim of having an effect on cysteine catabolism and showed promising results. NSCLC cell lines presented different cysteine metabolic patterns, with A549 and H292 presenting a higher reliance on cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) to maintain H2S levels, while the PC-9 cell line presented an adaptive behavior based on the use of mercaptopyruvate sulfurtransferase (MST) and cysteine dioxygenase (CDO1), both contributing to the role of cysteine as a pyruvate source. The analyses of human lung tumor samples corroborated this variability in profiles, meaning that the expression of certain genes may be informative in defining prognosis and new targets. Heterogeneity points out individual profiles, and the identification of new targets among metabolic players is a step forward in cancer management toward personalized medicine.

Tn and Sialyl-Tn antigens in canine gastric tissues.

Malignant transformation is often associated with abnormal protein glycosylation expressed, amongst others, by the accumulation of simple mucin-type carbohydrates namely Tn and Sialyl-Tn (STn) antigens. These are usually limited in normal tissues and their increased expression has been associated with cancer progression and poor prognosis. This study aims to evaluate the role of Tn and STn antigens in the neoplastic transformation of the canine gastric mucosa and to correlate their putative immunoexpression alterations with some pathological features. Tn and STn antigens expression were immunohistochemically evaluated in canine normal gastric mucosa (n = 3), gastric polyps (n = 9) and gastric carcinomas (n = 25), neoplastic emboli (n = 12) and metastases (n = 8). In normal gastric mucosa, Tn antigen was detected in the gastric epithelial cells, while STn antigen was absent. Similarly, all gastric polyps expressed Tn antigen, but none displayed STn antigen immunostaining. In carcinomas, Tn antigen was expressed in 96% of the cases and STn antigen in 68% of the neoplasms. STn antigen was significantly higher in carcinomas compared with normal mucosa (P < .05). No correlation was found between each antigen and the different subtypes of tumours according to WHO classification, tumour differentiation, lymph vascular invasion or metastasis. All neoplastic emboli expressed both antigens, and the expression score was similar or higher than that displayed by the neoplastic cells of the primary tumour. The high prevalence of STn antigen in gastric carcinomas compared with normal mucosa highlights the cancer-associated nature of this antigen. Our results link STn antigen expression to neoplastic transformation and suggest that it may be a useful marker of gastric cancer progression in dogs.

Sustained Auditory Attention Ability Test (SAAAT) in seven-year-old children with cleft lip and palate.

UNLABELLED: Cleft lip and palate (CLP) is a risk indicator to middle ear alterations, which may damage the development of auditory abilities such as attention that is essential to learn new skills, oral and written communication. Studies on attention process with CLP population are recent and poorly explored in the specific literature. Thus, this study aims to contribute with new subsidies in the field as it investigates the performance of children with CLP in Sustained Auditory Attention Ability Test (SAAAT). MATERIAL AND METHOD: Comparison of SAAAT performance between children with CLP and children without it. Prospective study. RESULTS: ANOVA was used as variance analysis model with two factors to study the variables such as group and gender. The CLP group showed an average of 2.5 units higher than the control group. This difference is between 0.7 and 4.4 with 95% certain. CONCLUSION: Children with cleft lip and palate had poorer performance on SAAAT when compared to those without such craniofacial anomaly, considering attention reduction only.

Teste de Habilidade de Atenção Auditiva Sustentada (THAAS) em crianças de sete anos com fissura labiopalatina / Sustained Auditory Attention Ability Test (SAAAT) in seven-year-old children with cleft lip and palate

A fissura labiopalatina é um indicador de risco para alterações de orelha média, o que pode prejudicar o desenvolvimento de habilidades auditivas tais como a atenção, que é essencial para o aprendizado de novas habilidades, comunicação oral e escrita. O estudo do processo atencional na população com fissura labiopalatina é algo recente e pouco explorado na literatura específica consultada, assim, este trabalho poderá contribuir com novos subsídios na área, uma vez que tem como objetivo verificar o desempenho de crianças com fissura labiopalatina no Teste de Habilidade de Atenção Auditiva Sustentada (THAAS). MATERIAL E MÉTODO: Comparação do desempenho no THAAS de crianças com e sem fissura labiopalatina. Estudo Prospectivo. RESULTADOS: Para estudar a associação entre os resultados do THAAS e as variáveis grupo e gênero foi utilizado um modelo de análise de variância (ANOVA) com dois fatores, com o qual foi possível observar que o grupo com fissura labiopalatina apresentou média 2,5 unidades maior que o grupo controle, esta diferença encontra-se entre 0,7 e 4,4 com 95 por cento de confiança. CONCLUSÃO: As crianças com fissura labiopalatina apresentaram desempenho no THAAS inferior àquelas sem essa anomalia craniofacial apenas para o decréscimo da vigilância.

Cleft lip and palate (CLP) is a risk indicator to middle ear alterations, which may damage the development of auditory abilities such as attention that is essential to learn new skills, oral and written communication. Studies on attention process with CLP population are recent and poorly explored in the specific literature. Thus, this study aims to contribute with new subsidies in the field as it investigates the performance of children with CLP in Sustained Auditory Attention Ability Test (SAAAT). MATERIAL AND METHOD: Comparison of SAAAT performance between children with CLP and children without it. Prospective study. RESULTS: ANOVA was used as variance analysis model with two factors to study the variables such as group and gender. The CLP group showed an average of 2.5 units higher than the control group. This difference is between 0.7 and 4.4 with 95 percent certain. CONCLUSION: children with cleft lip and palate had poorer performance on SAAAT when compared to those without such craniofacial anomaly, considering attention reduction only.

Frequency modulation (FM) system in auditory processing disorder: an evidence-based practice?

BACKGROUND: use of frequency modulation (FM) system in auditory processing disorder. AIM: to verify the existence of scientific evidence confirming the effectiveness of personal FM systems in the treatment of central auditory processing disorders (APD). For this purpose a systematic review of the literature was made, using data found in electronic databases (Medline, Lilacs and Cochrane library) as well as on the internet. The articles retrieved were analyzed according to the CONSORT statement and then classified by their evidence level. CONCLUSION: the search resulted in 1,589 references out of which only 19 met the inclusion criteria. All of the analyzed articles were classified as having low level of evidence (expert opinion or case studies). Strong scientific evidence supporting the use of personal FM systems for APD intervention was not found. Since such device is frequently recommended for the treatment of APD, it becomes essential to carry out studies with high scientific evidence that could safely guide clinical decision making on this subject.

Dichotic listening test (directed attention mode) in children with cleft lip and palate.

UNLABELLED: Conductive hearing loss in the first years of life may lead to hearing processing and attention deficit disorders, and consequently to communication and learning impairments. AIM: this paper aims to examine the performance of children with cleft lip and palate in dichotic listening tests (directed attention mode) and compare them to a control group without cleft lip and palate. MATERIALS AND METHOD: fifty-two children of both genders were enrolled in the study. Their ages ranged between 7 years and 7 years and 11 months, and they were divided into two groups: a study group featuring children with cleft lip and palate (n=27) and a control group with children without this anomaly (n=25). The children were first interviewed, then underwent a series of conventional hearing tests, and lastly were applied the dichotic hearing test. RESULTS: when submitted to the dichotic listening test (directed attention mode), the children in the study group had lower scores for both ears when compared to those in the control group. Statistical significance was found for variable gender in the groups, with p=0.026. CONCLUSION: in the dichotic listening test only the girls with cleft lip and palate had lower scores than the girls in the control group. This is a prospective clinical study.

Verbal recognition of infants with cleft lip and palate with and without history of risk indicators for hearing loss.

UNLABELLED: The first two years of life are critical for the acquisition and development of hearing and speaking skills. AIM: This prospective study aims to verify the performance of infants with cleft lip and palate (CLP) with and without risk factors for hearing (RFH) in the verbal recognition test (VRT). MATERIAL AND METHOD: The parents of 100 infants (9 to 18 months of age) with CLP were interviewed to investigate the presence of RFH and to sort out the characteristics of the study groups. All infants underwent VRT. RESULTS: Otologic diseases, lack of breastfeeding, parental smoking, upper airway insufficiency, stay in an incubator, and family history of hearing impairment were the most frequent RFH. Eighty-five infants had RFH, among which 40% had altered VRT results; fifteen did not have any RFH and 73% performed as expected for their age range in the VRT. There was no significant difference (p=0.326) between groups. Fifty-four infants had history of otitis media (OM), among which 31% had altered VRT results; forty-six had no history of OM and performed as expected for their age range in the VRT; Statistically significant difference (p=0.000) was found. CONCLUSION: Other risk factors for hearing aside CLP were found. Infants with and without history of RFH performed similarly in the VRT. The presence of otologic diseases significantly interfered with the VRT.

Teste Dicótico de Dígitos (etapa de escuta direcionada) em crianças com fissura labiopalatina / Dichotic Listening Test (directed attention mode) in children with cleft lip and palate

Perdas auditivas condutivas nos primeiros anos de vida podem levar a transtornos do processamento auditivo, de atenção e, conseqüentemente, dificuldades de aprendizado da comunicação. OBJETIVO: Verificar o desempenho de crianças com fissura labiopalatina no teste dicótico de dígitos, etapa de escuta direcionada; e compará-los aos de um grupo sem fissura labiopalatina. MATERIAL E MÉTODO: Fizeram parte deste estudo 52 crianças, de ambos os gêneros, na faixa etária de sete anos a sete anos e 11 meses, que foram distribuídas em dois grupos: um com crianças com fissura labiopalatina (n=27) e outro de crianças sem essa anomalia (n=25). O processo de avaliação constituiu da aplicação de um questionário, bateria de testes auditivos convencionais e aplicação do Teste Dicótico de Dígitos (etapa de escuta direcionada). RESULTADOS: No Teste Dicótico de Dígitos (etapa de escuta direcionada), foi possível observar que o grupo com FLP apresentou porcentagens de acerto inferiores ao grupo controle, tanto para a orelha direita quanto para a orelha esquerda. A análise estatística mostrou interação estatisticamente significativa para grupo versus gênero, p=0,026. CONCLUSÃO: No Teste Dicótico de Dígitos (etapa de escuta direcionada) somente as crianças do gênero feminino com fissura labiopalatina obtiveram índices de acerto inferiores às do grupo controle. Estudo prospectivo clínico.

Conductive hearing loss in the first years of life may lead to hearing processing and attention deficit disorders, and consequently to communication and learning impairments. AIM: this paper aims to examine the performance of children with cleft lip and palate in dichotic listening tests (directed attention mode) and compare them to a control group without cleft lip and palate. MATERIALS AND METHOD: fifty-two children of both genders were enrolled in the study. Their ages ranged between 7 years and 7 years and 11 months, and they were divided into two groups: a study group featuring children with cleft lip and palate (n=27) and a control group with children without this anomaly (n=25). The children were first interviewed, then underwent a series of conventional hearing tests, and lastly were applied the dichotic hearing test. RESULTS: when submitted to the dichotic listening test (directed attention mode), the children in the study group had lower scores for both ears when compared to those in the control group. Statistical significance was found for variable gender in the groups, with p=0.026. CONCLUSION: in the dichotic listening test only the girls with cleft lip and palate had lower scores than the girls in the control group. This is a prospective clinical study.

Avaliação do nível mínimo de audição em lactentes de seis a 24 meses por meio do reforço visual / Measurement of the minimal hearing level in infants from six to 24 months through visual reinforcement

OBJETIVO: Investigar, em campo livre, o comportamento auditivo de lactentes ouvintes de seis a 24 meses de idade cronológica, quanto aos aspectos de duração da avaliação, nível mínimo de resposta e análise crítica do método utilizado. MÉTODOS: Foram avaliados 30 lactentes, de ambos os gêneros, subdivididos em grupos etários: Grupo I - 6 meses a 11 meses 29 dias (n= 11); Grupo II - 12 meses a 17 meses 29 dias (n= 9); Grupo III - 18 meses a 24 meses (n= 10). Foram avaliadas as freqüências de 500, 1000, 2000 e 4000 Hz, bilateralmente, não seqüenciais, em campo livre. RESULTADOS: A mediana do nível mínimo de audição foi de 30 dB em todas as freqüências e todos os grupos. Observou-se que os Grupos I e III tiveram maior variabilidade dos níveis mínimos de audição enquanto o Grupo II mostrou-se mais homogêneo. O Grupo I necessitou de maior tempo de condicionamento, apresentando cansaço, agitação e tempo de atenção reduzido sendo necessário maior número de interrupções no exame. Já o Grupo III evidenciou menor motivação para responder ao teste, já que a técnica do reforço visual não é tão interessante nessa faixa etária, fazendo com que o desinteresse pelo reforço aconteça mais rapidamente. Estes aspectos não inviabilizam o procedimento, no entanto requerem maior experiência dos avaliadores. CONCLUSÕES: O método mostrou-se eficiente para a faixa etária pesquisada, não somente como um método de triagem, mas também para auxiliar a investigação do nível mínimo de audição, em campo livre, durante o processo diagnóstico.

PURPOSE: To investigate the auditory behavior of hearing infants with ages ranging from six to 24 months, on the aspects of evaluation length, minimum level of response and critical analysis of the method. METHODS: Thirty infants of both genders were divided in three age groups: Group I - 11 infants aged 6 to 11 months 29 days; Group II - 9 infants aged 12 to 17 months 29 days; Group III - 10 infants aged 18 to 24 months. Frequencies of 500, 1000, 2000 and 4000 Hz were bilaterally, non-consecutively evaluated in free field. RESULTS: The median of the minimum auditory level was 30 dB in all frequencies and groups. Groups I and III showed higher variability of minimum auditory levels, while Group II was more consistent. Group I needed more time to be conditioned, showing tiredness, excitement and short attention span, resulting in more interruptions during the test. Group III was less motivated to respond, since the technique of visual reinforcement is not so interesting at this age range, which makes the reinforcement even less exciting. These aspects do not make the procedure inadequate; however they require more experienced examiners. CONCLUSIONS: The method was efficient to the studied age range, not only as a screening procedure but also as a method to help the investigation of the minimum auditory level, in free field, during diagnostic process.

Atenção auditiva em crianças: estudo comparativo entre escolas públicas e privadas / Auditory awareness in children: a comparative study between public and private schools

Objetivo: comparar o desempenho de crianças de escolas públicas com as de escolas privadas, no teste de atenção auditiva FC2. Método: 154 crianças voluntárias de ambos os gêneros, com faixa etária de 6 a 11 anos foram avaliadas. As crianças foram divididas igualmente em 2 grupos: G1: estudantes de escola pública e G2: estudantes de escola privada. A avaliação constituiu-se da aplicação do teste de atenção auditiva FC2, que serve para avaliar a atenção auditiva verificando a habilidade de escutar estímulos auditivos durante um período de tempo prolongado e responder somente para um estímulo específico. Resultados: As crianças freqüentadoras de escola pública apresentaram escore mais rebaixado quando comparadas às de escola particular. Houve diferença estatisticamente significativa entre os dois grupos para as idades de 6, 8 e 9 anos para diferentes variáveis. Conclusão: A idade e o tipo de escola freqüentada pela criança devem ser considerados na aplicação do teste de atenção auditiva FC2.

Purpose: to compare the performance of private and public school children on auditory attention test FC2. Method: 154 volunteer children, of both genders, aged 6 to 11 years old were evaluated. The children were divided equally in two groups: G1: students of public schools and G2: students of private schools. The evaluation consisted of application of the test of auditory attention FC2, which evaluates the auditory attention verifying the ability to listen to auditory stimulus during a period of drawn out time and, to only answer for specific stimulus. Results: The children of public school showed score poorer than the ones of private school. There was statistically significant difference in performance between the groups to ages 6, 8 and 9 in different variables. Conclusion: The age and type of school the child goes must be taken into consideration for the application of FC2 test.