Repurposing discarded leukodepletion filters as a source of mononuclear cells for advanced in vitro research.

In light of advancements in the field of immuno-oncology, the demand for obtaining mononuclear cells for in vitro assays has surged. However, obtaining these cells from healthy donors remains a challenging task due to difficulties in donor recruitment and the requirement for substantial blood volumes. Here, we present a protocol for isolating peripheral blood mononuclear cells (PBMCs) from leukodepletion filters used in whole blood and erythrocytes by apheresis donations at the Hemonucleus of the Barretos Cancer Hospital, Brazil. The method involves rinsing the leukodepletion filters and subsequent centrifugation using a Ficoll-Paque concentration gradient. The isolated PBMCs were analyzed by flow cytometry, which allowed the identification of various subpopulations, including CD4+ T lymphocytes (CD45+CD4+), CD8+ T lymphocytes (CD45+CD8+), B lymphocytes (CD45+CD20+CD19+), non-classical monocytes (CD45+CD64+CD14-), classical monocytes (CD45+CD64+CD14+), and granulocytes (CD45+CD15+CD14-). In our comparative analysis of filters, we observed a higher yield of PBMCs from whole blood filters than those obtained from erythrocytes through apheresis. Additionally, fresh samples exhibited superior viability when compared to cryopreserved ones. Given this, leukodepletion filters provide a practical and cost-effective means to isolate large quantities of pure PBMCs, making it a feasible source for obtaining mononuclear cells for in vitro experiments. SUMMARY: Here, we provide a detailed protocol for the isolation of mononuclear cells from leukodepletion filters, which are routinely discarded at the Barretos Cancer Hospital's Hemonucleus.

Network meta-analysis of pembrolizumab as monotherapy and in combination with chemotherapy for first-line treatment of recurrent or metastatic head and neck squamous cell carcinoma / Metanálise em rede comparativa de pembrolizumabe como monoterapia e em combinação com quimioterapia para tratamento de primeira linha de carcinoma de células escamosas da cabeça e pescoço recorrente ou metastático

Pembrolizumab monotherapy or in combination with chemotherapy is approved as first-line treatment in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) based on improved overall survival (OS) versus EXTREME regimen in the KEYNOTE-048 trial. The clinical outcomes of pembrolizumab were compared with other recommended first-line treatments in R/M HNSCC in this study through a Bayesian network meta-analysis. A systematic literature review was conducted in July 2022, from which six trials that matched the KEYNOTE-048 patient eligibility criteria were included in the network. The OS and progression-free survival (PFS) outcomes were compared in the approved pembrolizumab indication (i.e., total population for pembrolizumab in combination with chemotherapy and combined positive score [CPS] ≥ 1 population for pembrolizumab monotherapy). A significant OS improvement was observed for pembrolizumab in combination with chemotherapy and pembrolizumab monotherapy versus EXTREME regimen (hazard ratio, 95% credible interval: 0.72, 0.60-0.86; 0.73, 0.60-0.88), platinum+5- FU (0.58, 0.43-0.76; 0.58, 0.44-0.78), and platinum+paclitaxel (0.53, 0.35-0.79; 0.53, 0.35-0.81), respectively. A non-significant numeric trend in OS improvement was observed versus the TPEx regimen. PFS was comparable with most first-line treatments and was improved versus platinum+5-FU (0.48, 0.36-0.64; 0.59, 0.45-0.79). Additional analyses in higher CPS subgroups also showed consistent results. Overall, our study results showed an improvement in OS outcomes versus alternative first-line treatments, consistent with the findings of the KEYNOTE-048 trial. These data support using pembrolizumab as a suitable firstline treatment option in R/M HNSCC.

Pembrolizumabe em monoterapia ou em combinação com quimioterapia é aprovado como tratamento de primeira linha em carcinoma de células escamosas recorrente/metastático de cabeça e pescoço (CECCP R/M) com base na melhora da sobrevida global (OS), em comparação com o esquema EXTREME no estudo KEYNOTE-048. Esse estudo comparou os resultados clínicos de pembrolizumabe com outros tratamentos recomendados de primeira linha em CECCP R/M por meio de uma metanálise de rede bayesiana. Uma revisão sistemática da literatura foi conduzida em julho de 2022, a partir da qual seis ensaios clínicos que atendiam aos critérios de elegibilidade de pacientes do KEYNOTE-048 foram incluídos na rede. Os desfechos de OS e sobrevida livre de progressão (PFS) foram comparados na indicação de pembrolizumabe (população total para pembrolizumabe em combinação com quimioterapia e população com escore positivo combinado [CPS] ≥ 1 em monoterapia com pembrolizumabe). Foi observada melhora significativa na OS para pembrolizumabe em combinação com quimioterapia e monoterapia com pembrolizumabe versus o esquema EXTREME (razão de risco, intervalo de confiança de 95%: 0,72, 0,60-0,86; 0,73, 0,60-0,88), platina+5-FU (0,58, 0,43-0,76; 0,58, 0,44-0,78) e platina+paclitaxel (0,53, 0,35-0,79; 0,53, 0,35-0,81), respectivamente. Uma tendência numérica não significativa de melhoria na OS foi observada em relação ao esquema TPEx. A PFS foi comparável com a maioria dos tratamentos de primeira linha e melhor em relação à platina+5-FU (0,48, 0,36-0,64; 0,59, 0,45-0,79). Análises adicionais em subgrupos com CPS mais elevado também mostraram resultados consistentes. No geral, os resultados de nosso estudo mostraram melhora nos desfechos de OS em comparação aos tratamentos de primeira linha alternativos, consistentes com os achados do estudo KEYNOTE-048. Esses dados apoiam o uso de pembrolizumabe como opção de tratamento em primeira linha em pacientes com CECCP R/M.

Systemic treatment in patients with Child-Pugh B liver dysfunction and advanced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of death among patients with liver cirrhosis. The rise of immuno-oncology has revolutionized treatment for advanced HCC. However, most pivotal randomized controlled trials have excluded patients with moderate liver dysfunction (Child-Pugh-Turcotte B), despite the high incidence of liver disease in patients with HCC at the time of diagnosis. Overall survival in patients with HCC and moderate liver dysfunction treated with sorafenib has been found to be only approximately 3-5 months, underlining the need for improved treatment algorithms for this increasingly important subgroup of patients. In this review, we summarize available data on the treatment of patients with HCC and moderate liver dysfunction. Opportunities, as well as clinical challenges, are discussed in detail, highlighting potential changes to the therapeutic landscape.

Novel evaluation approach for molecular signature-based deconvolution methods.

The tumoral immune microenvironment (TIME) plays a key role in prognosis, therapeutic approach and pathophysiological understanding over oncological processes. Several computational immune cell-type deconvolution methods (DM), supported by diverse molecular signatures (MS), have been developed to uncover such TIME interplay from RNA-seq tumor biopsies. MS-DM pairs were benchmarked against each other by means of different metrics, such as Pearson's correlation, R2 and RMSE, but these only evaluate the linear association of the estimated proportion related to the expected one, missing the analysis of prediction-dependent bias trends and cell identification accuracy. We present a novel protocol composed of four tests allowing appropriate evaluation of the cell type identification performance and proportion prediction accuracy of molecular signature-deconvolution method pair by means of certainty and confidence cell-type identification scores (F1-score, distance to the optimal point and error rates) as well the Bland-Altman method for error-trend analysis. Our protocol was used to benchmark six state-of-the-art DMs (CIBERSORTx, DCQ, DeconRNASeq, EPIC, MIXTURE and quanTIseq) paired to five murine tissue-specific MSs, revealing a systematic overestimation of the number of different cell types across almost all methods.

Immuno-oncology in head and neck squamous cell carcinoma - a narrative review

Immuno-oncology studies the immune system in cancer. In recent decades, immunotherapy has shown a good response to the treatment of various locally advanced and metastatic cancers. The main mechanisms of action include stimulation of the patient's own immune system to enhance immune responses acting in tumor escape pathways. This review examined the literature related to immune system mechanisms in head and neck squamous cell carcinoma (HNSCC) and their application in immunotherapy using biomarkers. The PUBMED, LILACS, MEDLINE, WHOLIS, and SCIELO databases were searched using the terms squamous cell carcinoma, head and neck, immuno-oncology, immunotherapy, and immunology. The main drugs currently available for clinical use in patients diagnosed with HNSCC include pembrolizumab and nivolumab, both classified as check-point inhibitors. These immunobiological agents improve patient survival and quality of life. Many authors and clinical trials point out that the recommendation of these agents is linked to the dose of PD-L1 (ligand expressed primarily by tumor cells), which proved to be an unreliable biomarker in the patient selection. Recommendation of immunotherapy depends on reliable biomarkers that must be identified in order to achieve good therapeutic results.

Comprehensive Characterization of the Regulatory Landscape of Adrenocortical Carcinoma: Novel Transcription Factors and Targets Associated with Prognosis.

We reconstructed a transcriptional regulatory network for adrenocortical carcinoma (ACC) using transcriptomic and clinical data from The Cancer Genome Atlas (TCGA)-ACC cohort. We investigated the association of transcriptional regulatory units (regulons) with overall survival, molecular phenotypes, and immune signatures. We annotated the ACC regulons with cancer hallmarks and assessed single sample regulon activities in the European Network for the Study of Adrenal Tumors (ENSAT) cohort. We found 369 regulons associated with overall survival and subdivided them into four clusters: RC1 and RC2, associated with good prognosis, and RC3 and RC4, associated with worse outcomes. The RC1 and RC3 regulons were highly correlated with the 'Steroid Phenotype,' while the RC2 and RC4 regulons were highly correlated with a molecular proliferation signature. We selected two regulons, NR5A1 (steroidogenic factor 1, SF-1) and CENPA (Centromeric Protein A), that were consistently associated with overall survival for further downstream analyses. The CENPA regulon was the primary regulator of MKI-67 (a marker of proliferation KI-67), while the NR5A1 regulon is a well-described transcription factor (TF) in ACC tumorigenesis. We also found that the ZBTB4 (Zinc finger and BTB domain-containing protein 4) regulon, which is negatively associated with CENPA in our transcriptional regulatory network, is also a druggable anti-tumorigenic TF. We anticipate that the ACC regulons may be used as a reference for further investigations concerning the complex molecular interactions in ACC tumors.

Leveraging NKG2D Ligands in Immuno-Oncology.

Immune checkpoint inhibitors (ICI) revolutionized the field of immuno-oncology and opened new avenues towards the development of novel assets to achieve durable immune control of cancer. Yet, the presence of tumor immune evasion mechanisms represents a challenge for the development of efficient treatment options. Therefore, combination therapies are taking the center of the stage in immuno-oncology. Such combination therapies should boost anti-tumor immune responses and/or target tumor immune escape mechanisms, especially those created by major players in the tumor microenvironment (TME) such as tumor-associated macrophages (TAM). Natural killer (NK) cells were recently positioned at the forefront of many immunotherapy strategies, and several new approaches are being designed to fully exploit NK cell antitumor potential. One of the most relevant NK cell-activating receptors is NKG2D, a receptor that recognizes 8 different NKG2D ligands (NKG2DL), including MICA and MICB. MICA and MICB are poorly expressed on normal cells but become upregulated on the surface of damaged, transformed or infected cells as a result of post-transcriptional or post-translational mechanisms and intracellular pathways. Their engagement of NKG2D triggers NK cell effector functions. Also, MICA/B are polymorphic and such polymorphism affects functional responses through regulation of their cell-surface expression, intracellular trafficking, shedding of soluble immunosuppressive isoforms, or the affinity of NKG2D interaction. Although immunotherapeutic approaches that target the NKG2D-NKG2DL axis are under investigation, several tumor immune escape mechanisms account for reduced cell surface expression of NKG2DL and contribute to tumor immune escape. Also, NKG2DL polymorphism determines functional NKG2D-dependent responses, thus representing an additional challenge for leveraging NKG2DL in immuno-oncology. In this review, we discuss strategies to boost MICA/B expression and/or inhibit their shedding and propose that combination strategies that target MICA/B with antibodies and strategies aimed at promoting their upregulation on tumor cells or at reprograming TAM into pro-inflammatory macrophages and remodeling of the TME, emerge as frontrunners in immuno-oncology because they may unleash the antitumor effector functions of NK cells and cytotoxic CD8 T cells (CTL). Pursuing several of these pipelines might lead to innovative modalities of immunotherapy for the treatment of a wide range of cancer patients.

First-line Treatment of Metastatic Renal Cell Carcinoma in the Immuno-oncology Era: Systematic Review and Network Meta-analysis.

Combination treatments with immuno-oncology (IO) agents and IO agents plus a vascular endothelial growth factor receptor tyrosine kinase inhibitor (VEGFR-TKI) have been approved for first-line treatment of patients with metastatic renal cell carcinoma (mRCC). No direct comparisons have been performed among these treatment options. We performed a systematic review and network meta-analysis to compare and rank the available regimens for first-line treatment in terms of survival benefit and efficacy. In accordance with the Preferred Reporting Items for Systematic Review statement, a systematic search of reported studies was performed in MEDLINE, the Cochrane Central Register of Controlled Trials, and EMBASE up to May 31, 2019. Network meta-analysis models were adjusted using the Bayesian method. Four randomized clinical trials, with a total of 3758 patients, met the inclusion criteria. Considering systemic therapy, 1880 patients had received sunitinib and 550, 432, 442, and 454 patients had received ipilimumab plus nivolumab (ipi + nivo), pembrolizumab plus axitinib (pembro + axi), avelumab plus axitinib (avelu + axi), and atezolizumab plus bevacizumab (atezo + bev). No difference was found in overall survival between ipi + nivo and pembro + axi for the intention to treat population (hazard ratio [HR], 1.34; 95% credible interval [CrI], 0.92-1.97). No difference was found in progression-free survival among the treatments. The overall response rate (ORR) was superior with pembro + axi and avelu + axi compared with the ORR with the other treatments (atezo + bev vs. pembro + axi: HR, 0.66; 95% CrI, 0.52-0.84; ipi + nivo vs. pembro + axi: HR, 0.73; 95% CrI, 0.59-0.90; atezo + bev vs. avelu + axi: HR, 0.55; 95% CrI, 0.43-0.71; avelu + axi vs. ipi + nivo: HR, 1.66; 95% CrI, 1.31-2.12), with no differences across them (HR, 1.21; 95% CrI, 0.95-1.53). In the present indirect comparison, for an intention to treat population, we found no survival differences between pembro + axi and ipi + nivo. All treatments showed better progression-free survival compared with sunitinib that was similar among them. The combination of an IO agent (pembrolizumab or avelumab) and axitinib seemed to be the most effective therapy for the ORR.

LAG-3 antagonists by cancer treatment: a patent review.

Introduction: LAG-3 is checkpoint inhibitor in cancer that coordinates the downregulation of the proliferation of antigen-specific lymphocytes. There is a great need to discover and develop new therapies focused on inhibiting the action of LAG-3 and consequently improving the immune response in the various types of cancer. Areas covered: The patent literature reveals novel therapies, which provide information on cancer therapies. The authors used the patent databases of the six main patent offices of the world: United States Patent and Trademark Office, European Patent Office, World Intellectual Property Organization, Japan Patent Office, State Intellectual Property Office of China and Korean Intellectual Property Office, to generate a detailed landscape of patents and patent applications of active companies related to LAG-3 inhibitors. Specific patents have been grouped into innovative patents and adopting patents. Expert opinion: There is a continuing development of LAG-3 inhibitors, and these inhibitors are being used in combination with other cancer treatment schemes, for example, antibodies against PD-1, PD-L1, and CTLA-4. Immutep and IO Therapeutics were the leaders in generating innovator patents, followed by Gustave Roussy Institute, and Applied Research Systems ARS. Dana-Farber Cancer Institute was the leader in the generation of adopter patents, followed by Novartis .

Cancer immunotherapy using anti-TIM3/PD-1 bispecific antibody: a patent evaluation of EP3356411A1.

Introduction: TIM3 and PD-1 are checkpoint inhibitors in cancer that coordinate the downregulation of the proliferation of antigen-specific lymphocytes. There is a great need to discover and develop new therapies focused on inhibiting the action of TIM3 and PD-1 and consequently improving the immune response in the various types of cancer. The authors of patent EP3356411A1 propose several anti-TIM3/anti-PD-1 bispecific antibodies, as well as the method for producing them and their pharmacological application in the treatment of cancer. Areas covered: Patent EP3356411A1 describes a method by producing anti-TIM3/anti-PD-1 bispecific antibodies and their potential in cancer treatment. Expert opinion: Data supporting the patent demonstrate the ability by producing anti-TIM3/anti-PD-1 bispecific antibodies. Although the proposed methodology is very interesting and promising, further studies are necessary to assess the clinical applicability of the inventions on cancer.

The Value of Biomarkers in Optimizing the Use of Immuno-oncologic Therapy.

The development of therapies that restore or activate the host immune response - the socalled "immuno-oncologic" therapy - has improved the survival of some cancer patients harboring specific tumor types. These drugs, however, are very expensive which has greatly limited their use and consequently reduced the number of patients who could likely benefit. Not to mention, the proportion of patients who display a clinical benefit from therapy is limited. Thus, from a clinical and health economics perspective, there is a pressing need to identify and treat those patients for whom a given immuno- oncologic therapy is most likely to be beneficial. At this end, the identification, validation and use of biomarkers emerge as an important therapeutic tool. Here, we briefly review the state of immunologic biomarker development and utilization and make suggestions for interested clinicians, health policy makers and other stakeholders to prepare for the broader use of biomarkers associated with immuno-oncologic therapy in routine practice. The biomarker field is clearly in its earliest stages and there is no doubt that continued research will identify new biomarkers with valuable clinical indications. Of course, the clinical utility of a biomarker must consider patient preferences and perspectives. In addition, health economic analyses are crucial to better define the value of immunotherapy based on precision medicine strategies and promote value-based pricing.

Thromboembolic Events Related to Treatment with Checkpoint Inhibitors: Report of Two Cases.

Despite the significant clinical benefits, checkpoint inhibition is associated with a unique spectrum of immune-related adverse events. It is sometimes difficult to distinguish some rare adverse effects from a cancer progression; thus, such effects should be reported in clinical trials to be diagnosed by physicians. Only a few cases of arterial embolic events have been described in studies related to patients treated by immunotherapy. In this article, we report the cases of 2 patients who presented rare and severe thromboembolic events after using checkpoint inhibitors. The first case describes multiple organ embolism at the same time, associated with other autoimmune symptoms. In the second case, distal digital necrosis emerged after the initiation of immunotherapy. There is insufficient data about the real incidence of thromboembolic and rheumatological events related to checkpoint inhibition. Future trials should be done to establish preventive strategies.

Custo-efetividade de terapias anti-PD-1 comparadas com ipilimumabe para o tratamento de melanoma avançado sob a perspectiva do sistema de saúde suplementar brasileiro / Cost-effectiveness of anti-PD-1 therapies compared with ipilimumab for the treatment of advanced melanoma under the Brazilian supplementary health system perspective

Objetivo: Realizar uma análise de custo-efetividade das terapias imuno-oncológicas anti-PD-1 aprovadas no Brasil versus ipilimumabe no tratamento do paciente sem tratamento prévio com melanoma metastático (estádios III ou IV), independentemente da mutação BRAF sob a perspectiva do sistema de saúde suplementar brasileiro. Métodos: Foi desenvolvido um modelo com três estados de saúde mutuamente exclusivos (livre de progressão, progressão da doença e morte) para simular a condição clínica de pacientes tratados com nivolumabe ou pembrolizumabe comparado ao ipilimumabe. Os custos de medicamentos, materiais, exames e procedimentos foram calculados com base na lista oficial de preços no Brasil ­ CMED (março/2017), revistas Kairos e Simpro, Planserv 2008 e CBHPM 2015. O desfecho clínico considerado para a análise foi de anos de vida salvos. Resultados: O nivolumabe produziu uma razão de custo-efetividade incremental de R$ 37.231 e o pembrolizumabe, de R$ 72.760. Ambas as intervenções demonstraram benefício clínico dentro do limiar de disposição a pagar recomendado pela OMS (três vezes o PIB per capita), mostrando que as tecnologias são custoefetivas. Na análise de sensibilidade univariada foi demonstrado que as RCEIs para ambas as análises foram mais sensíveis aos parâmetros referentes à taxa de desconto anual e aos custos de acompanhamento. Entre as terapias imuno-oncológicas anti-PD-1, o nivolumabe apresentou benefício clínico maior a um custo menor. Conclusão: Ambas as terapias anti-PD-1 (nivolumabe e pembrolizumabe) são custo-efetivas versus ipilimumabe, sugerindo-se o nivolumabe como melhor opção para a alocação de recursos no tratamento de pacientes sem tratamento prévio com melanoma avançado, independentemente da mutação BRAF, sob a perspectiva do sistema de saúde suplementar brasileiro.

Objective: The aim of this study was to evaluate the cost-effectiveness of anti-PD-1 therapies approved in Brazil versus ipilimumab for the treatment of previously untreated patients with metastatic melanoma (stage III/IV) irrespective of BRAF status under the Brazilian supplementary health system perspective. Methods: A cost-effectiveness model with three mutually exclusive health state (pre-progression, post-progression and death) was developed to simulate the clinical condition of patients with metastatic melanoma treated with nivolumab or pembrolizumab compared with ipilimumab. The cost of drugs, materials, exams and procedures were obtained from official Brazilian price list ­ CMED, Kairos and Simpro magazines, Planserv 2008 and CBHPM 2015. The clinical outcome considered in the analysis was life years saved. Results: Nivolumab produced an incremental cost-effectiveness ratio of R$ 37,231 and pembrolizumab of R$ 72,760. Both interventions offered clinical benefit within the willingness-to-pay threshold recommended by World Health Organization (WHO) (three times per-capita GDP), showing that the technologies are cost-effective. It was demonstrated in the univariate sensitivity analyses that the parameters in which ICER of the comparison of nivolumab vs. ipilimumab and pembrolizumab vs. ipilimumab were more sensitive to annual discount rate (costs) and follow-up costs. Conclusion: Both nivolumab and pembrolizumab are cost-effective versus ipilimumab, suggesting that it would be more willing to be adopted for the treatment of previously untreated patients with advanced melanoma regardless of BRAF mutation under Brazilian supplementary health system perspective.