Not Only RET but NF1 and Chromosomal Instability Are Seen in Young Patients with Sporadic Medullary Thyroid Carcinoma.

Context: Genetic analysis of sporadic medullary thyroid carcinoma (MTC) has revealed somatic variants in RET, RAS, and occasionally other genes. However, around 20% of patients with sporadic MTC lack a known genetic driver. Objective: To uncover potential new somatic or germline drivers, we analyze a distinct cohort of patients with sporadic, very early-onset, and aggressive MTC. Methods: Germline and somatic DNA exome sequencing was performed in 19 patients, previously tested negative for germline RET variants. Results: Exome sequencing of 19 germline samples confirmed the absence of RET and identified an NF1 pathogenic variant in 1 patient. Somatic sequencing was successful in 15 tumors revealing RET variants in 80%, predominantly p.Met918Thr, which was associated with disease aggressiveness. In RET-negative tumors, pathogenic variants were found in HRAS and NF1. The NF1 germline and somatic variants were observed in a patient without a prior clinical diagnosis of neurofibromatosis type 1, demonstrating that the loss of heterozygosity of NF1 functions as a potential MTC driver. Somatic copy number alterations analysis revealed chromosomal alterations in 53.3% of tumors, predominantly in RET-positive cases, with losses in chromosomes 9 and 22 being the most prevalent. Conclusion: This study reveals that within a cohort of early-onset nonhereditary MTC, RET remains the major driver gene. In RET-negative tumors, NF1 and RAS are drivers of sporadic MTC. In addition, in young patients without a RET germline mutation, a careful clinical evaluation with a consideration of germline NF1 gene analysis is ideal to exclude Neurofibromatosis type 1 (NF1).

A systematic review of clinical trials for gene therapies for ß-hemoglobinopathy around the world.

BACKGROUND AIMS: Amidst the success of cell therapy for the treatment of onco-hematological diseases, the first recently Food and Drug Administration-approved gene therapy product for patients with transfusion-dependent ß-thalassemia (TDT) indicates the feasibility of gene therapy as curative for genetic hematologic disorders. This work analyzed the current-world scenario of clinical trials involving gene therapy for ß-hemoglobinopathies. METHODS: Eighteen trials for patients with sickle cell disease (SCD) and 24 for patients with TDT were analyzed. RESULTS: Most are phase 1 and 2 trials, funded by the industry and are currently recruiting volunteers. Treatment strategies for both diseases are fetal hemoglobin induction (52.4%); addition of wild-type or therapeutic ß-globin gene (38.1%) and correction of mutations (9,5%). Gene editing (52.4%) and gene addition (40.5%) are the two most used techniques. The United States and France are the countries with the greatest number of clinical trials centers for SCD, with 83.1% and 4.2%, respectively. The United States (41.1%), China (26%) and Italy (6.8%) lead TDT trials centers. CONCLUSIONS: Geographic trial concentration indicates the high costs of this technology, logistical issues and social challenges that need to be overcome for gene therapy to reach low- and middle-income countries where SCD and TDT are prevalent and where they most impact the patient's health.

Systematic Review of Available CAR-T Cell Trials around the World.

In this systematic review, we foresee what could be the approved scenario in the next few years for CAR-T cell therapies directed against hematological and solid tumor malignancies. China and the USA are the leading regions in numbers of clinical studies involving CAR-T. Hematological antigens CD19 and BCMA are the most targeted, followed by mesothelin, GPC3, CEA, MUC1, HER2, and EGFR for solid tumors. Most CAR constructs are second-generation, although third and fourth generations are being largely explored. Moreover, the benefit of combining CAR-T treatment with immune checkpoint inhibitors and other drugs is also being assessed. Data regarding product formulation and administration, such as cell phenotype, transfection technique, and cell dosage, are scarce and could not be retrieved. Better tracking of trials' status and results on the ClinicalTrials.gov database should aid in a more concise and general view of the ongoing clinical trials involving CAR-T cell therapy.

COVID-19 impact on anxiety and depression in head and neck cancer patients: A cross-sectional study.

OBJECTIVE: To evaluate whether the coronavirus disease 2019 has increased anxiety, depression, and distress levels in head and neck cancer (HNC) patients undergoing radiotherapy (RT). METHODS: In this cross-sectional study, RT-HNC patients were surveyed using the Hospital Anxiety and Depression Scale (HADS) for anxiety and depression and the distress thermometer (DT) for distress. HADS scores were compared with data pre-COVID-19. Additionally, we evaluated the COVID-19 impact on daily routines, treatment, and cancer care through a questionnaire. RESULTS: Fifty patients were included. The HADS mean score and estimated rates were 4.34 (±4.06)/22% for anxiety and 5.08 (±4.82)/22% for depression; in comparison, our historical control had 4.04 (±3.59)/20% for anxiety (p = .79) and 4.03 (±3.62)/17% for depression (p = .49). Mean DT score was 3.68 (±2.77). Responders were aware of COVID-19, afraid of having medical complications, believed it was life-threatening, did not miss appointments, believed their treatment was not impacted, and felt safe at the hospital amid the pandemic. CONCLUSION: This study suggests that anxiety, depression, and distress levels found in RT-HNC patients did not increase during the pandemic. Patients were afraid of being infected by COVID-19; however, they complied with their cancer treatment.

In Search of an Ideal CAR-T Cell Antigen Target.

Chimeric antigen receptor T (CAR-T) cells are proving their value in hematological cancers such as B cell acute lymphoid leukemia (B-ALL). This success is in great part due to the chosen target antigen, the lineage marker CD19, that is expressed by leukemia blasts and B lymphocytes, which are not crucial. For solid tumors, the challenge is greater because antigen expression is highly heterogeneous within the tumor and even an efficient CAR-T strategy would not kill all tumor cells. Also, many antigens are shared between solid tumors and healthy cells, causing off-target cell lysis and dangerous collateral damage. New antigen sources are emerging as targets, such as viruses, endogenous viruses, and immune checkpoint molecules. New technologies are in search of the ideal target, with antigen combinations the leading candidates.

CRISPR / Cas-9 Vector System: Targets UL-39 and Inhibits HSV-1 Replication in Vitro

HSV-1 affects approximately 67% of the world population. Here, we sought to use the CRISPR / Cas9 system with the UL39 target, essential for virus replication. The sgRNA sequence was inserted into plasmid (PX459-UL39). Vero cells were transfected with PX459-UL39, and inhibition of viral replication was assessed 24 and 48 hours later using plaque assays and fluorescence and qPCR. Fluorescence analyzes revealed the presence of anti-HSV-1 CRISPR/Cas9 within Vero cells, and qPCR showed that the viral load decreased by> 95% of cells transfected with anti-HSV-1 CRISPR / Cas9. Our data demonstrate the usefulness of the PX459-UL39 to inhibit HSV-1 infection.

High infiltration of B cells in tertiary lymphoid structures, TCR oligoclonality, and neoantigens are part of esophageal squamous cell carcinoma microenvironment.

Esophageal squamous cell carcinoma (ESCA) exhibits high intratumoral molecular heterogeneity posing a challenge to cancer therapy. Immune checkpoint blockade therapy has been approved for this disease, but with modest results. RNA-Seq data from paired tumor and surrounding nonmalignant tissue from 14 patients diagnosed with ESCA without previous treatment and from The Cancer Genome Atlas-ESCA cohort were analyzed. Herein, we investigated ESCA immune landscape including mutation-derived neoantigens and immune cell subpopulations. Tumor-associated antigen expression was determined by in silico analyses and confirmed by immunohistochemistry showing that PRAME, CEACAM4, and MAGEA11 proteins are expressed on tumors. Immune checkpoint molecules gene expression was higher in the tumor compared with surrounding nonmalignant tissue, but its expression varies greatly among patients. TCR repertoire and BCR transcripts analysis evidenced low clonal diversity with one TCR clone predicted to be specific for a MAGEA11-derived peptide. A high number of B-cell clones infiltrating the tumors and the abundance of these cells in tertiary lymphoid structures observed in ESCA tumors support B cells as a potential immune modulator in this tumor.

Distinct pattern of one-carbon metabolism, a nutrient-sensitive pathway, in invasive breast cancer: A metabolomic study.

Altered cell metabolism is a hallmark of cancer and critical for its development. Particularly, activation of one-carbon metabolism in tumor cells can sustain oncogenesis while contributing to epigenetic changes and metabolic adaptation during tumor progression. We assessed whether increased one-carbon metabolism activity is a metabolic feature of invasive ductal carcinoma (IDC). Differences in the metabolic profile between biopsies from IDC (n = 47) and its adjacent tissue (n = 43) and between biopsies from different breast cancer subtypes were assessed by gas spectrometry in targeted (Biocrates Life Science ® ) and untargeted approaches, respectively. The metabolomics data were statistically treated using MetaboAnalyst 4.0, SIMCA P+ (version 12.01), Statistica 10 software and t test with p < 0.05. The Cancer Genome Atlas breast cancer dataset was also assessed to validate the metabolomic profile of IDC. Our targeted metabolomics analysis showed distinct metabolomics profiles between IDC and adjacent tissue, where IDC displayed a comparative enrichment of metabolites involved in one-carbon metabolism (serine, glycine, threonine, and methionine) and a predicted increase in the activity of pathways that receive and donate carbon units (i.e., folate, methionine, and homocysteine). In addition, the targeted and untargeted metabolomics analyses showed similar metabolomics profiles between breast cancer subtypes. The gene set enrichment analysis identified different transcription-related functions between IDC and non-tumor tissues that involved one-carbon metabolism. Our data suggest that one-carbon metabolism may be a central pathway in IDC and even in general breast tumors, representing a potential target for its treatment and prevention.

Development of CAR-T cell therapy for B-ALL using a point-of-care approach.

Recently approved by the FDA and European Medicines Agency, CAR-T cell therapy is a new treatment option for B-cell malignancies. Currently, CAR-T cells are manufactured in centralized facilities and face bottlenecks like complex scaling up, high costs, and logistic operations. These difficulties are mainly related to the use of viral vectors and the requirement to expand CAR-T cells to reach the therapeutic dose. In this paper, by using Sleeping Beauty-mediated genetic modification delivered by electroporation, we show that CAR-T cells can be generated and used without the need for ex vivo activation and expansion, consistent with a point-of-care (POC) approach. Our results show that minimally manipulated CAR-T cells are effective in vivo against RS4;11 leukemia cells engrafted in NSG mice even when inoculated after only 4 h of gene transfer. In an effort to better characterize the infused CAR-T cells, we show that 19BBz T lymphocytes infused after 24 h of electroporation (where CAR expression is already detectable) can improve the overall survival and reduce tumor burden in organs of mice engrafted with RS4;11 or Nalm-6 B cell leukemia. A side-by-side comparison of POC approach with a conventional 8-day expansion protocol using Transact beads demonstrated that both approaches have equivalent antitumor activity in vivo. Our data suggest that POC approach is a viable alternative for the generation and use of CAR-T cells, overcoming the limitations of current manufacturing protocols. Its use has the potential to expand CAR immunotherapy to a higher number of patients, especially in the context of low-income countries.

4T1 Mammary Carcinoma Colonization of Metastatic Niches Is Accelerated by Obesity.

Breast cancer (BC) remains the leading cause of cancer-related deaths among women, and the chances to develop it are duplicated by obesity. Still, the impact of obesity during BC progression remains less understood. We investigated the role of obesity in tumor progression using the murine model of 4T1 mammary carcinoma in BALB/c female mice, previously high-fat-diet (HFD) fed. HFD induced obesity, metabolic impairment, and high serum and fat leptin levels. After injection of 4T1-cells, HFD-mice accelerated tumor progression and metastasis. 4T1-cells found within HFD-mice metastatic niches presented higher clonogenic potential. 4T1-cells treated in vitro with fat-conditioned medium derived from HFD-mice, increased migration capacity through CXCL12 and CCL25 gradients. In HFD-mice, the infiltration and activation of immune cells into tumor-sentinel lymph nodes was overall reduced, except for activated CD4+ T cells expressing low CD25 levels. Within the bone marrow, the levels of haematopoiesis-related IL-6 and TNF-α decreased after 4T1-cells injection in HFD-mice whereas increased in the controls, suggesting that upregulation of both cytokines, regardless of the tumor, is disrupted by obesity. Finally, the expression of genes for leptin, CXCR4, and CCR9 (receptors of CXCL12 and CCL25, respectively) was negatively correlated with the infiltration of CD8 T cells in human triple-negative BC tumors from obese patients compared to non-obese. Together, our data present early evidence of systemic networks triggered by obesity that promote BC progression to the metastatic niches. Targeting these pathways might be useful to prevent the rapid BC progression observed among obese patients.

CAR T Cells Generated Using Sleeping Beauty Transposon Vectors and Expanded with an EBV-Transformed Lymphoblastoid Cell Line Display Antitumor Activity In Vitro and In Vivo.

Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of cancer is now an approved treatment for B cell malignancies. However, the use of viral vectors to provide long-term CAR expression is associated with high production costs and cumbersome quality controls, impacting the final cost of CAR T cell therapies. Nonviral integrative vectors, such as Sleeping Beauty (SB) transposons, provide an alternative to modify primary T cells. Therefore, we developed a protocol to expand SB-transfected 19BBζ CAR T cells using a lymphoblastoid cell line, and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased cell viability on day 1 but had a minor impact on the frequency of memory subpopulations when compared to mock condition. CAR+ lymphocytes showed increased proliferation compared to mock control and high cytotoxic activity towards CD19+ cells without significant differences in exhaustion markers expression. Moreover, CAR+ lymphocytes showed an increased frequency by the end of the stimulation cycle compared with day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NOD scid gamma chain knockout (NSG) mice engrafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBζ CAR T cells showed improved overall survival when compared to mock T cells treated animals. The results showed that electroporation using the SB system is a simple and affordable method for inducing long-term CAR expression in T lymphocytes. Expansion of gene-modified T cells with the lymphoblastoid cell line provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.

Transcriptional characterization of immunological infiltrates and their relation with glioblastoma patients overall survival.

Introduction: Several cell populations from the peripheral immune system interact to create a complex immunologic status during glioblastoma growth and response to therapy. The aim of this study was to integrate the impact of different immune cell populations present in glioblastoma tumor microenvironment on overall survival. Methodology: Gene expression and clinical data were generated by The Cancer Genome Atlas and previously reported meta-signatures representing cells of the immune system were used. The relationship between meta-signatures was evaluated through Pearson's correlation analyses. Survival analyses were performed through Kaplan-Meier plots and Cox regression model. Results and discussion: Meta-signatures corresponding to infiltrating immune cells with immunosuppressive roles, such as macrophages, NK and NK T cells, MDSCs and Tregs, correlated with poorer patient prognosis. Meta-signatures related to CD8+ T cells predicted improved survival only in patients with low immunosuppressive meta-signatures. By clustering the meta-signatures we found that the cluster containing high meta-signatures of macrophages, MDSCs and Tregs demonstrated the worst prognosis. Conclusion: Integrating the information provided by transcriptional signatures of immunological aspects is fundamental in understanding the impact of the immune system on patient survival. We found a predictive impact on survival with positive role for CD8 and negative roles for macrophages, MDSC, Tregs, NK and NK-T in glioblastoma patients. Understanding these regulatory and stimulatory factors of patients' immune system is essential to delineate an effective strategy to increase the anti-tumor immune response and to generate potential clinical benefits.

Células epiteliais do timo são possível reservatório viral e transmitem o HTLV-1 para linfócitos T CD4 / Thymic epithelial cells are infected by htlv-1 and transmit the virus to cd4 t lymphocytes

O timo é um órgão linfoide primário, sítio do desenvolvimento de células T, provendofatores críticos e coordenados que induzem e suportam o comprometimento delinhagem, diferenciação e sobrevivência dessas células. A presença das célulasnão-linfóides, principalmente as células epiteliais do timo (TEC) no parênquimatímico promove a migração e diferenciação coordenada dos linfócitos T. Os linfócitosT são o principal alvo do Virus linfotrópico T humano (HTLV-1), agente etiológico daleucemia/linfoma associado ao HTLV-1 (ATL) e de doença que compromete osistema nervoso/muscular (HAM/TSP). É desconhecida a causa que leva a uma ououtra doença. A resposta imune antiviral mediada por células T é ineficiente nessaspatologias. Mesmo que o vírus tenha tropismo pelos linfócitos T, ele é capaz deinfectar outros tipos celulares por contato direto entre células ou por partículas viraislivres. Linfócitos ativados recirculam pelos órgãos linfoides, incluindo o timo, onde ascélulas epiteliais tímicas (TEC) interagem intimamente com as células recirculantes,promovendo uma possível via de transmição do HTLV-1. No nosso trabalho,observamos que as TECs possuem os receptores para a entrada do vírus (GLUT-1e Neuropilina-1). Experimentos in vitro mostraram que as TECs podem serinfectadas pelo HTLV-1 por linhagens de linfócitos derivados de pacientesportadores de ATL e de HAM/TSP. Essas infecções ocorreram tanto por contatodireto entre as células, quanto por sobrenadante contendo partículas virais livresderivadas do sobrenadante dos linfócitos. O vírus pode ser observado após 24horas e 10 dias de cultivo, quando a maioria das células estava infectada. Atravésde microscopia eletrônica de transmissão, foram observadas partículas viraisbrotando de estruturas semelhantes a corpos multivesculares nas TECs...

The thymus is a primary lymphoid organ, site of developing T cells, providing acoordinated set of critical factors to induce and support lineage commitment,differentiation and survival of these cells. The presence of non-lymphoid cellsthrough the thymic parenchyma serves to provide coordinated migration anddifferentiation of T lymphocytes. T lymphocytes are the main target of Human TLymphotropic Virus type 1 (HTLV-1). This virus is the etiologic agent of HTLV-1associated lymphoma and leukemia (ATL) or a disease of muscular/nervous systems(HAM/TSP), but it is unknown what triggers to one or other disease. T-cell mediatedimmune response against viral proteins is not effective in both diseases. Althoughthe virus has T lymphocyte tropism, it can infect other cells by cell contact and cellfreevirus. Activated T lymphocytes circulates around lymphoid organs, including thethymus, where the thymic epithelial cells (TEC) strongly interact with recirculatingcells, so infected lymphocytes could transmit the virus to TEC. Interestingly, in ourwork we observed that TEC expresses molecules related to the HTLV-1 transmission(GLUT-1, Neuropilin 1). In vitro experiments showed that TEC could be infected bycell lineages derived from ATL and HAM/TSP patients. These infections happenedfrom cell contact and by cell-free virus derived from cell supernatants. The virus canbe seen after 24h and after 10 days, when most cells in the culture were infected. Bytransmission electron microscopy, virus particles were observed budding frommultivesicular bodies like structures in TEC. Cytokines and chemokines wereincreased at mRNA level soon after contact with HTLV-1 containing supernatantderived from lymphocytes. In addition, type 1 interferon and interferon stimulatedgenes were decreased in HTLV-1 infected TEC...