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1.
J Transl Med ; 22(1): 270, 2024 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-38475820

RESUMO

Most anti-cancer modalities are designed to directly kill cancer cells deploying mechanisms of action (MOAs) centered on the presence of a precise target on cancer cells. The efficacy of these approaches is limited because the rapidly evolving genetics of neoplasia swiftly circumvents the MOA generating therapy-resistant cancer cell clones. Other modalities engage endogenous anti-cancer mechanisms by activating the multi-cellular network (MCN) surrounding neoplastic cells in the tumor microenvironment (TME). These modalities hold a better chance of success because they activate numerous types of immune effector cells that deploy distinct cytotoxic MOAs. This in turn decreases the chance of developing treatment-resistance. Engagement of the MCN can be attained through activation of immune effector cells that in turn kill cancer cells or when direct cancer killing is complemented by the production of proinflammatory factors that secondarily recruit and activate immune effector cells. For instance, adoptive cell therapy (ACT) supplements cancer cell killing with the release of homeostatic and pro-inflammatory cytokines by the immune cells and damage associated molecular patterns (DAMPs) by dying cancer cells. The latter phenomenon, referred to as immunogenic cell death (ICD), results in an exponential escalation of anti-cancer MOAs at the tumor site. Other approaches can also induce exponential cancer killing by engaging the MCN of the TME through the release of DAMPs and additional pro-inflammatory factors by dying cancer cells. In this commentary, we will review the basic principles that support emerging paradigms likely to significantly improve the efficacy of anti-cancer therapy.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Neoplasias/terapia , Antineoplásicos/uso terapêutico , Citocinas/metabolismo , Microambiente Tumoral
2.
Int J Mol Sci ; 25(17)2024 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-39273294

RESUMO

Resistance biomarkers are needed to identify patients with advanced melanoma obtaining a response to ICI treatment and developing resistance later. We searched a combination of molecular signatures of response to ICIs in patients with metastatic melanoma. In a retrospective study on patients with metastatic melanoma treated with an anti-PD-1 agent carried out at Istituto Nazionale Tumori-IRCCS-Fondazione "G. Pascale", Naples, Italy. We integrated a whole proteome profiling of metastatic tissue with targeted transcriptomics. To assess the prognosis of patients according to groups of low and high risk, we used PFS and OS as outcomes. To identify the proteins and mRNAs gene signatures associated with the patient's response groups, the discriminant analysis for sparse data performed via partial least squares procedure was performed. Tissue samples from 22 patients were analyzed. A combined protein and gene signature associated with poorer response to ICI immunotherapy in terms of PFS and OS was identified. The PFS and OS Kaplan-Meier curves were significantly better for patients with high expression of the protein signature compared to patients with low expression of the protein signature and who were high-risk (Protein: HR = 0.023, 95% CI: 0.003-0.213; p < 0.0001. Gene: HR = 0.053, 95% CI: 0.011-0.260; p < 0.0001). The Kaplan-Meier curves showed that patients with low-risk gene signatures had better PFS (HR = 0 0.221, 95% CI: 0.071-0.68; p = 0.007) and OS (HR = 0.186, 95% CI: 0.05-0.695; p = 0.005). The proteomic and transcriptomic combined analysis was significantly associated with the outcomes of the anti-PD-1 treatment with a better predictive value compared to a single signature. All the patients with low expression of protein and gene signatures had progression within 6 months of treatment (median PFS = 3 months, 95% CI: 2-3), with a significant difference vs. the low-risk group (median PFS = not reached; p < 0.0001), and significantly poorer survival (OS = 9 months, 95% CI: 5-9) compared to patients with high expression of protein and gene signatures (median OS = not reached; p < 0.0001). We propose a combined proteomic and transcriptomic signature, including genes involved in pro-tumorigenic pathways, thereby identifying patients with reduced probability of response to immunotherapy with ICIs for metastatic melanoma.


Assuntos
Inibidores de Checkpoint Imunológico , Melanoma , Proteômica , Transcriptoma , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Melanoma/metabolismo , Melanoma/mortalidade , Feminino , Masculino , Estudos Retrospectivos , Proteômica/métodos , Pessoa de Meia-Idade , Inibidores de Checkpoint Imunológico/uso terapêutico , Idoso , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/genética , Biomarcadores Tumorais/genética , Adulto , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteoma/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/metabolismo , Metástase Neoplásica
3.
J Transl Med ; 21(1): 610, 2023 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-37684649

RESUMO

BACKGROUND: Identifying response markers is highly needed to guide the treatment strategy in patients with metastatic melanoma. METHODS: A retrospective study was carried out in patients with unresectable/metastatic melanoma (stage IIIb-IV), treated with anti-PD-1 in the first line setting, to better explore the role and the timing of neutrophil/lymphocyte ratio (NLR) as potential biomarker of response. The relationship of NLR with inflammation-immune mediators and the underlying negative effect of raising NLR during immunotherapy, have been investigated with transcriptomic gene analysis. RESULTS: The results confirmed previous findings that a high baseline NLR is associated with a poorer prognosis and with higher serum level of lactate dehydrogenase (LDH), regardless of the presence of brain metastases. The transcriptomic analysis showed that high baseline NLR is associated with a characteristic gene signature CCNA1, LDHA and IL18R1, which correlates with inflammation and tumorigenesis. Conversely, low baseline NLR is associated with the signature CD3, SH2D1A, ZAP70 and CD45RA, linked to the immune-activation. The genes positively associated with NLR (CD39 (ENTPD1), PTEN, MYD88, MMP9 and LDH) are involved in processes of immunosuppression, inflammation and tumor-promoting activity. Increased expression of CD39 correlated with TGFß2, a marker of the N2 neutrophils with immunosuppressive activity. CONCLUSIONS: These results suggest that increasing NLR is associated with an increased neutrophil population, with polarization to the N2 phenotype, and this process may be the basis for the negatively prognostic role of NLR.


Assuntos
Melanoma , Neutrófilos , Humanos , Prognóstico , Estudos Retrospectivos , Imunoterapia , Melanoma/genética , Melanoma/terapia
4.
J Transl Med ; 19(1): 459, 2021 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-34743703

RESUMO

BACKGROUND: Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. METHODS: To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. RESULTS: Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product. CONCLUSIONS: As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.


Assuntos
Neoplasias , Anticorpos de Cadeia Única , Antígenos CD28/genética , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva , RNA Guia de Cinetoplastídeos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T
5.
Haematologica ; 106(9): 2405-2416, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817282

RESUMO

Recent randomized trials focused on gene expression-based determination of the cell of origin in diffuse large B-cell lymphoma could not show significant improvements by adding novel agents to standard chemoimmunotherapy. The aim of this study was the identification of a gene signature able to refine current prognostication algorithms and applicable to clinical practice. Here we used a targeted gene expression profiling panel combining the Lymph2Cx signature for cell of origin classification with additional targets including MYC, BCL-2 and NFKBIA, in 186 patients from 2 randomized trials (discovery cohort) (NCT00355199 and NCT00499018). Data were validated in 3 independent series (2 large public datasets and a real-life cohort). By integrating the cell of origin, MYC/BCL-2 double expressor status and NFKBIA expression, we defined a 3-gene signature combining MYC, BCL-2 and NFKBIA (MBN-signature), which outperformed the MYC/BCL-2 double expressor status in multivariate analysis, and allowed further risk stratification within the germinal center B-cell/unclassified subset. The high-risk (MBN Sig-high) subgroup identified the vast majority of double hit cases and a significant fraction of Activated B-Cell-derived diffuse large B-cell lymphomas. These results were validated in 3 independent series including a cohort from the REMoDL-B trial, where, in an exploratory ad hoc analysis, the addition of bortezomib in the MBN Sig-high subgroup provided a progression free survival advantage compared with standard chemoimmunotherapy. These data indicate that a simple 3-gene signature based on MYC, BCL-2 and NFKBIA could refine the prognostic stratification in diffuse large B-cell lymphoma, and might be the basis for future precision-therapy approaches.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Linfoma Difuso de Grandes Células B , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Perfilação da Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Inibidor de NF-kappaB alfa , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-myc/genética , Medição de Risco
6.
Cancer Treat Res ; 180: 149-172, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32215869

RESUMO

The development of cancer results from the evolutionary balance between the proliferating aptitude of cancer cells and the response of the host's tissues. Some cancers are characterized by genetic instability dependent upon impaired DNA repair mechanisms that lead to the chaotic disruption of multiple cellular functions often in excess of the cancer survival needs and may exert broad effects on surrounding tissues, some beneficial and some detrimental to cancer growth. Among them, inflammatory processes that accompany wound healing may initiate a reaction of the host against the neo-formation. This is possibly triggered by the release by dying cancer cells of molecules known as Damage-Associated Molecular Patterns (DAMPs) following a process termed Immunogenic Cell Death (ICD) that initiates an immune response through innate and adaptive mechanisms. Indeed, analysis of large cancer data sets has shown that ICD is strictly associated with the activation of other immune effector or immune-regulatory pathways. Here, we will describe how immune activation and compensatory immune-regulatory mechanisms balance anti-cancer immune surveillance and the roles that innate and adaptive immunity play including the weight that neo-epitopes may exert as initiators and sculptors of high-affinity memory and effector immune responses against cancer. We will discuss the evolutionary basis for the existence of immune checkpoints and how several theories raised to explain cancer resistance to immunotherapy represent a facet of a similar evolutionary phenomenon that we described in the Theory of Everything. We will show how the biology of immunogenicity and counterbalancing immune regulation is widespread across cancers independent of their ontogenesis while subtle idiosyncratic differences are discernible among them. Finally, we will suggest that overcoming immune resistance implies distinct approaches relevant to the immune context of individual cancers.


Assuntos
Morte Celular Imunogênica , Neoplasias/imunologia , Imunidade Adaptativa , Alarminas/imunologia , Antígenos de Neoplasias/imunologia , Epitopos , Humanos , Imunidade Inata , Imunoterapia
7.
Cancer Treat Res ; 180: 173-195, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32215870

RESUMO

Checkpoint inhibitor therapy (CIT) has revolutionized cancer treatment but it has also reached a standstill when an absent dialog between cancer and immune cells makes it irrelevant. This occurs with high prevalence in the context of "immune silent" and, even perhaps, "immune-excluded" tumors. The latter are characterized by T cells restricted to the periphery of cancer nests. Since in either case T cells do not come in direct contact with most cancer cells, CIT rests immaterial. Adoptive cell therapy (ACT), may also be affected by limited access to antigen-bearing cancer cells. While lack of immunogenicity intuitively explains the immune silent phenotype, immune exclusion is perplexing. The presence of T cells at the periphery suggests that chemo-attraction recruits them and an immunogenic stimulus promotes their persistence. However, what stops the T cells from infiltrating the tumors' nests and reaching the germinal center (GC)? Possibly, a concentric gradient of increased chemo-repulsion or decreased chemo-attraction demarcates an abrupt "do not trespass" warning. Various hypotheses suggest physical or functional barriers but no definitive consensus exists over the weight that each plays in human cancers. On one hand, it could be hypothesized that the intrinsic biology of cancer cells may degenerate from a "cancer stem cell" (CSC)-like phenotype in the GC toward a progressively more immunogenic phenotype prone to immunogenic cell death (ICD) at the periphery. On the other hand, the intrinsic biology of the cancer cells may not change but it is the disorderly architecture of the tumor microenvironment (TME) that alters in a centripetal direction cancer cell metabolism, both directly and indirectly, the function of surrounding stromal cells. In this chapter, we examine whether the paradoxical exclusion of T cells from tumors may serve as a model to understand the requirements for tumor immune infiltration and, correspondingly, we put forth strategies to restore the dialog between immune cells and cancer to enhance the effectiveness of immune oncology (IO) approaches.


Assuntos
Neoplasias/imunologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Humanos , Morte Celular Imunogênica , Imunoterapia Adotiva
8.
J Transl Med ; 17(1): 357, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31684954

RESUMO

BACKGROUND: The 18-gene tumor inflammation signature (TIS) is a clinical research assay that enriches for clinical benefit to immune checkpoint blockade. We evaluated its ability to predict clinical benefit of immunotherapy in cancer patients treated with PD-1 checkpoint inhibitors in routine clinical care. METHODS: The CERTIM cohort is a prospective cohort which includes patients receiving immune checkpoint inhibitors in Cochin University hospital. RNA extracted from 58 archival formalin fixed paraffin embedded tumor blocks (including 38 lung cancers, 5 melanomas, 10 renal carcinomas, 4 urothelial carcinomas and 1 colon carcinoma) was hybridized to a beta version of the NanoString® PanCancer IO360™ CodeSet using nCounter® technology. Gene expression signatures were correlated with tumor responses (by RECIST criteria) and overall survival. PD-L1 immunostaining on tumor cells was assessed in 37 non-small cell lung cancer (NSCLC) samples and tumor mutational burden (TMB) measured by whole exome sequencing in 19 of these. RESULTS: TIS scores were significantly associated with complete or partial response to anti-PD-1 treatment in the whole cohort (odds ratio = 2.64, 95% CI [1.4; 6.0], p = 0.008), as well as in the NSCLC population (odds ratio = 3.27, 95% CI [1.2; 11.6], p = 0.03). Patients whose tumor had a high TIS score (upper tertile) showed prolonged overall survival compared to patients whose tumor had lower TIS scores, both in the whole cohort (hazard ratio = 0.37, 95% CI [0.18, 0.76], p = 0.005) and in the NSCLC population (hazard ratio = 0.36, 95% CI [0.14, 0.90], p = 0.02). In the latter, the TIS score was independent from either PD-L1 staining on tumor cells (spearman coefficient 0.2) and TMB (spearman coefficient - 0.2). CONCLUSIONS: These results indicate that validated gene expression assay measuring the level of tumor microenvironment inflammation such as TIS, are accurate and independent predictive biomarkers and can be easily implemented in the clinical practice.


Assuntos
Inflamação/genética , Inflamação/terapia , Neoplasias/genética , Neoplasias/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Estudos de Coortes , Feminino , Humanos , Imunoterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Mutação , Estudos Prospectivos , Transcriptoma , Pesquisa Translacional Biomédica , Resultado do Tratamento
9.
J Transl Med ; 15(1): 223, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29100546

RESUMO

Immunotherapies have emerged as one of the most promising approaches to treat patients with cancer. Recently, the entire medical oncology field has been revolutionized by the introduction of immune checkpoints inhibitors. Despite success in a variety of malignancies, responses typically only occur in a small percentage of patients for any given histology or treatment regimen. There are also concerns that immunotherapies are associated with immune-related toxicity as well as high costs. As such, identifying biomarkers to determine which patients are likely to derive clinical benefit from which immunotherapy and/or be susceptible to adverse side effects is a compelling clinical and social need. In addition, with several new immunotherapy agents in different phases of development, and approved therapeutics being tested in combination with a variety of different standard of care treatments, there is a requirement to stratify patients and select the most appropriate population in which to assess clinical efficacy. The opportunity to design parallel biomarkers studies that are integrated within key randomized clinical trials could be the ideal solution. Sample collection (fresh and/or archival tissue, PBMC, serum, plasma, stool, etc.) at specific points of treatment is important for evaluating possible biomarkers and studying the mechanisms of responsiveness, resistance, toxicity and relapse. This white paper proposes the creation of a network to facilitate the sharing and coordinating of samples from clinical trials to enable more in-depth analyses of correlative biomarkers than is currently possible and to assess the feasibilities, logistics, and collated interests. We propose a high standard of sample collection and storage as well as exchange of samples and knowledge through collaboration, and envisage how this could move forward using banked samples from completed studies together with prospective planning for ongoing and future clinical trials.


Assuntos
Biomarcadores Tumorais/metabolismo , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Humanos , Internacionalidade , Reprodutibilidade dos Testes , Estatística como Assunto
10.
J Transl Med ; 14(1): 313, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27846884

RESUMO

The sixth "Melanoma Bridge Meeting" took place in Naples, Italy, December 1st-4th, 2015. The four sessions at this meeting were focused on: (1) molecular and immune advances; (2) combination therapies; (3) news in immunotherapy; and 4) tumor microenvironment and biomarkers. Recent advances in tumor biology and immunology has led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS) of cancer patients. Immunotherapies in particular have emerged as highly successful approaches to treat patients with cancer including melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer, and Hodgkin's disease. Specifically, many clinical successes have been using checkpoint receptor blockade, including T cell inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death-1 (PD-1) and its ligand PD-L1. Despite demonstrated successes, responses to immunotherapy interventions occur only in a minority of patients. Attempts are being made to improve responses to immunotherapy by developing biomarkers. Optimizing biomarkers for immunotherapy could help properly select patients for treatment and help to monitor response, progression and resistance that are critical challenges for the immuno-oncology (IO) field. Importantly, biomarkers could help to design rational combination therapies. In addition, biomarkers may help to define mechanism of action of different agents, dose selection and to sequence drug combinations. However, biomarkers and assays development to guide cancer immunotherapy is highly challenging for several reasons: (i) multiplicity of immunotherapy agents with different mechanisms of action including immunotherapies that target activating and inhibitory T cell receptors (e.g., CTLA-4, PD-1, etc.); adoptive T cell therapies that include tissue infiltrating lymphocytes (TILs), chimeric antigen receptors (CARs), and T cell receptor (TCR) modified T cells; (ii) tumor heterogeneity including changes in antigenic profiles over time and location in individual patient; and (iii) a variety of immune-suppressive mechanisms in the tumor microenvironment (TME) including T regulatory cells (Treg), myeloid derived suppressor cells (MDSC) and immunosuppressive cytokines. In addition, complex interaction of tumor-immune system further increases the level of difficulties in the process of biomarkers development and their validation for clinical use. Recent clinical trial results have highlighted the potential for combination therapies that include immunomodulating agents such as anti-PD-1 and anti-CTLA-4. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors on T cells and other approaches such as adoptive cell transfer are tested for clinical efficacy in melanoma as well. These agents are also being tested in combination with targeted therapies to improve upon shorter-term responses thus far seen with targeted therapy. Various locoregional interventions that demonstrate promising results in treatment of advanced melanoma are also integrated with immunotherapy agents and the combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for melanoma patients' population. This meeting's specific focus was on advances in immunotherapy and combination therapy for melanoma. The importance of understanding of melanoma genomic background for development of novel therapies and biomarkers for clinical application to predict the treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into personalized-medicine approach for treatment of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma. We also discussed the requirements for pre-analytical and analytical as well as clinical validation process as applied to biomarkers for cancer immunotherapy. The concept of the fit-for-purpose marker validation has been introduced to address the challenges and strategies for analytical and clinical validation design for specific assays.


Assuntos
Pesquisa Biomédica , Melanoma/patologia , Animais , Biomarcadores Tumorais/metabolismo , Ensaios Clínicos como Assunto , Terapia Combinada , Humanos , Imunoterapia , Itália , Melanoma/genética , Melanoma/imunologia , Melanoma/terapia , Microambiente Tumoral
11.
J Transl Med ; 12: 184, 2014 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-24965603

RESUMO

BACKGROUND: Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity. METHODS: Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates. RESULTS: Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples. CONCLUSIONS: SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.


Assuntos
Dano ao DNA , Reparo do DNA , Análise de Célula Única/métodos , Adulto , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Criança , Ciclina A2/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Haploinsuficiência/efeitos dos fármacos , Histonas/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Humanos , Mutagênicos/toxicidade , Fosforilação/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Reprodutibilidade dos Testes , Temozolomida
12.
J Transl Med ; 12: 178, 2014 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-24952610

RESUMO

BACKGROUND: Single-cell network profiling (SCNP) is a multiparametric flow cytometry-based approach that simultaneously measures evoked signaling in multiple cell subsets. Previously, using the SCNP approach, age-associated immune signaling responses were identified in a cohort of 60 healthy donors. METHODS: In the current study, a high-dimensional analysis of intracellular signaling was performed by measuring 24 signaling nodes in 7 distinct immune cell subsets within PBMCs in an independent cohort of 174 healthy donors [144 elderly (>65 yrs); 30 young (25-40 yrs)]. RESULTS: Associations between age and 9 immune signaling responses identified in the previously published 60 donor cohort were confirmed in the current study. Furthermore, within the current study cohort, 48 additional immune signaling responses differed significantly between young and elderly donors. These associations spanned all profiled modulators and immune cell subsets. CONCLUSIONS: These results demonstrate that SCNP, a systems-based approach, can capture the complexity of the cellular mechanisms underlying immunological aging. Further, the confirmation of age associations in an independent donor cohort supports the use of SCNP as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies.


Assuntos
Envelhecimento/imunologia , Voluntários Saudáveis , Transdução de Sinais , Adulto , Idoso , Estudos de Coortes , Humanos
13.
J Immunol ; 188(4): 1717-25, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22246624

RESUMO

A greater understanding of the function of the human immune system at the single-cell level in healthy individuals is critical for discerning aberrant cellular behavior that occurs in settings such as autoimmunity, immunosenescence, and cancer. To achieve this goal, a systems-level approach capable of capturing the response of the interdependent immune cell types to external stimuli is required. In this study, an extensive characterization of signaling responses in multiple immune cell subpopulations within PBMCs from a cohort of 60 healthy donors was performed using single-cell network profiling (SCNP). SCNP is a multiparametric flow cytometry-based approach that enables the simultaneous measurement of basal and evoked signaling in multiple cell subsets within heterogeneous populations. In addition to establishing the interindividual degree of variation within a broad panel of immune signaling responses, the possible association of any observed variation with demographic variables including age and race was investigated. Using half of the donors as a training set, multiple age- and race-associated variations in signaling responses in discrete cell subsets were identified, and several were subsequently confirmed in the remaining samples (test set). Such associations may provide insight into age-related immune alterations associated with high infection rates and diminished protection following vaccination and into the basis for ethnic differences in autoimmune disease incidence and treatment response. SCNP allowed for the generation of a functional map of healthy immune cell signaling responses that can provide clinically relevant information regarding both the mechanisms underlying immune pathological conditions and the selection and effect of therapeutics.


Assuntos
Envelhecimento/imunologia , Negro ou Afro-Americano , Sistema Imunitário/metabolismo , Leucócitos Mononucleares/imunologia , Transdução de Sinais , Análise de Célula Única/métodos , População Branca , Adulto , Idoso , Doenças Autoimunes/imunologia , Células Cultivadas , Estudos de Coortes , Citocinas/biossíntese , Feminino , Citometria de Fluxo/métodos , Humanos , Sistema Imunitário/imunologia , Imunidade Celular , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia
14.
Br J Haematol ; 162(2): 250-62, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23682827

RESUMO

Single cell network profiling (SCNP) is a multi-parameter flow cytometry technique for simultaneous interrogation of intracellular signalling pathways. Diagnostic paediatric acute myeloid leukaemia (AML) bone marrow samples were used to develop a classifier for response to induction therapy in 53 samples and validated in an independent set of 68 samples. The area under the curve of a receiver operating characteristic curve (AUC(ROC)) was calculated to be 0·85 in the training set and after exclusion of induction deaths, the AUC(ROC) of the classifier was 0·70 (P = 0·02) and 0·67 (P = 0·04) in the validation set when induction deaths (intent to treat) were included. The highest predictive accuracy was noted in the cytogenetic intermediate risk patients (AUC(ROC) 0·88, P = 0·002), a subgroup that lacks prognostic/predictive biomarkers for induction response. Only white blood cell count and cytogenetic risk were associated with response to induction therapy in the validation set. After controlling for these variables, the SCNP classifier score was associated with complete remission (P = 0·017), indicating that the classifier provides information independent of other clinical variables that were jointly associated with response. This is the first validation of an SCNP classifier to predict response to induction chemotherapy. Herein we demonstrate the usefulness of quantitative SCNP under modulated conditions to provide independent information on AML disease biology and induction response.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Adolescente , Criança , Pré-Escolar , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Feminino , Citometria de Fluxo/métodos , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Terapia Neoadjuvante , Prognóstico , Estudos Prospectivos , Indução de Remissão , Estudos Retrospectivos , Análise de Célula Única/métodos , Tioguanina/administração & dosagem , Resultado do Tratamento
15.
J Transl Med ; 11: 137, 2013 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-23731854

RESUMO

Recent insights into the genetic and somatic aberrations have initiated a new era of rapidly evolving targeted and immune-based treatments for melanoma. After decades of unsuccessful attempts to finding a more effective cure in the treatment of melanoma now we have several drugs active in melanoma. The possibility to use these drugs in combination to improve responses to overcome the resistance, to potentiate the action of immune system with the new immunomodulating antibodies, and identification of biomarkers that can predict the response to a particular therapy represent new concepts and approaches in the clinical management of melanoma. The third "Melanoma Research: "A bridge from Naples to the World" meeting, shortened as "Bridge Melanoma Meeting" took place in Naples, December 2 to 4th, 2012. The four topics of discussion at this meeting were: advances in molecular profiling and novel biomarkers, combination therapies, novel concepts toward integrating biomarkers and therapies into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage, and the knowledge gained from the biology of tumor microenvironment across different tumors as a bridge to impact on prognosis and response to therapy in melanoma. This international congress gathered more than 30 international faculty members who in an interactive atmosphere which stimulated discussion and exchange of their experience regarding the most recent advances in research and clinical management of melanoma patients.


Assuntos
Melanoma/diagnóstico , Melanoma/terapia , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/terapia , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Ensaios Clínicos como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Oncologia/tendências , Melanoma/metabolismo , Mutação , Prognóstico
16.
Cytometry A ; 83(4): 386-95, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23300058

RESUMO

The aim of this study was to assess the feasibility of applying the single cell network profiling (SCNP) assay to the examination of signaling networks in epithelial cancer cells, using bladder washings from 29 bladder cancer (BC) and 15 nonbladder cancer (NC) subjects. This report describes the methods we developed to detect rare epithelial cells (within the cells we collected from bladder washings), distinguish cancer cells from normal epithelial cells, and reproducibly quantify signaling within these low frequency cancer cells. Specifically, antibodies against CD45, cytokeratin, EpCAM, and cleaved-PARP (cPARP) were used to differentiate nonapoptotic epithelial cells from leukocytes, while measurements of DNA content to determine aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK at baseline and in response to pathway modulation; 66% (N = 19) of BC samples and 27% (N = 4) of NC samples met the "evaluable" criteria, i.e., at least 400,000 total cells available upon sample receipt with >2% of cells showing an epithelial phenotype. The majority of epithelial cells detected in BC samples were nonapoptotic and all signaling data were generated from identified cPARP negative cells. In four of 19 BC samples but in none of the NC specimens, SCNP assay identified epithelial cancer cells with a quantifiable increase in epidermal growth factor-induced p-AKT and p-ERK levels. Furthermore, preincubation with the PI3K inhibitor GDC-0941 reduced or completely inhibited basal and epidermal growth factor-induced p-AKT but, as expected, had no effect on p-ERK levels. This study demonstrates the feasibility of applying SCNP assay using multiparametric flow cytometry to the functional characterization of rare, bladder cancer cells collected from bladder washing. Following assay standardization, this method could potentially serve as a tool for disease characterization and drug development in bladder cancer and other solid tumors.


Assuntos
Biomarcadores Tumorais/genética , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-akt/genética , Neoplasias da Bexiga Urinária/genética , Aneuploidia , Biomarcadores Tumorais/metabolismo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/classificação , Células Epiteliais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Citometria de Fluxo/métodos , Humanos , Indazóis/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Sulfonamidas/farmacologia , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
17.
Haematologica ; 98(4): 626-34, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23144194

RESUMO

While many prognostic markers in B-cell chronic lymphocytic leukemia provide insight into the biology of the disease, few have been demonstrated to be useful in the daily management of patients. B-cell receptor signaling is a driving event in the progression of B-cell chronic lymphocytic leukemia and markers of B-cell receptor responsiveness have been shown to be of prognostic value. Single cell network profiling, a multiparametric flow cytometry-based assay, allows functional signaling analysis at the level of the single cell. B-cell receptor signaling proteins (i.e. p-SYK, p-NF-κB p65, p-ERK, p-p38, p-JNK) were functionally characterized by single cell network profiling in samples from patients with B-cell chronic lymphocytic leukemia in an exploratory study (n=27) after stimulation with anti-IgM. Significant associations of single cell network profiling data with clinical outcome (i.e. time to first treatment), as assessed by Cox regression models, were then confirmed in patients' samples in two other sequential independent studies, i.e. test study 1 (n=30), and test study 2 (n=37). In the exploratory study, higher responsiveness of the B-cell receptor signaling proteins to anti-IgM was associated with poor clinical outcomes. Patients' clustering based on signaling response was at least as powerful in discriminating different disease courses as traditional prognostic markers. In an unselected subgroup of patients with Binet stage A disease (n=21), increased anti-IgM-modulated p-ERK signaling was shown to be a significant, independent predictor of shorter time to first treatment. This result was independently confirmed in two test cohorts from distinct populations of patients. In conclusion, these findings support the utility of the single cell network profiling assay in elucidating signaling perturbations with the potential for the development of a clinically useful prognostic test in patients with early stage B-cell chronic lymphocytic leukemia. These data support the clinical relevance of B-cell receptor signaling in B-cell chronic lymphocytic leukemia, and suggest a key role of ERK activation in the physiopathology of this leukemia.


Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Leucócitos Mononucleares/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Análise de Célula Única/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Anti-Idiotípicos/farmacologia , Células Cultivadas , Progressão da Doença , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Citometria de Fluxo/métodos , Citometria de Fluxo/estatística & dados numéricos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , NF-kappa B/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk
18.
J Transl Med ; 10: 113, 2012 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-22672557

RESUMO

BACKGROUND: Single-cell network profiling (SCNP) is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity. METHODS: The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs), 5 European Americans (36-56 yrs), all males]. RESULTS: Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NF-κB pathway (NF-κB). In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies. CONCLUSIONS: SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Understanding race-associated contrasts in immune cell signaling responses may be one critical component for elucidation of differences in immune-mediated disease prevalence and treatment responses.


Assuntos
Grupos Raciais , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Adulto , Negro ou Afro-Americano , Linfócitos B/imunologia , Citometria de Fluxo , Humanos , Imunoglobulina D/imunologia , Cinética , Masculino , Pessoa de Meia-Idade , Doadores de Tecidos , População Branca
19.
Haematologica ; 102(4): e144-e147, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27927769
20.
Blood Adv ; 6(15): 4634-4644, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35767735

RESUMO

T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) is a rare and aggressive variant of diffuse large B-cell lymphoma (DLBCL) that usually affects young to middle-aged patients, with disseminated disease at presentation. The tumor microenvironment (TME) plays a key role in THRLBCL due to its peculiar cellular composition (<10% neoplastic B cells interspersed in a cytotoxic T-cell/histiocyte-rich background). A significant percentage of THRLBCL is refractory to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP)-based regimens and to chimeric antigen receptor T-cell therapy; thus, the development of a specific therapeutic approach for these patients represents an unmet clinical need. To better understand the interaction of immune cells in THRLBCL TME and identify more promising therapeutic strategies, we compared the immune gene expression profiles of 12 THRLBCL and 10 DLBCL samples, and further corroborated our findings in an extended in silico set. Gene coexpression network analysis identified the predominant role of the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis in the modulation of the immune response. Furthermore, the PD-1/PD-L1 activation was flanked by the overexpression of 48 genes related to the functional exhaustion of T cells. Globally, THRLBCL TME was highly interferon-inflamed and severely exhausted. The immune gene profiling findings strongly suggest that THRLBCL may be responsive to anti-PD-1 therapy but also allowed us to take a step forward in understanding THRLBCL TME. Of therapeutic relevance, we validated our results by immunohistochemistry, identifying a subset of TCF1+ (T cell-specific transcription factor 1, encoded by the TCF7 gene) progenitor exhausted T cells enriched in patients with THRLBCL. This subset of TCF1+ exhausted T cells correlates with good clinical response to immune checkpoint therapy and may improve prediction of anti-PD-1 response in patients with THRLBCL.


Assuntos
Antígeno B7-H1 , Linfoma Difuso de Grandes Células B , Apoptose , Antígeno B7-H1/genética , Fator 1-alfa Nuclear de Hepatócito , Histiócitos/metabolismo , Histiócitos/patologia , Humanos , Ligantes , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Receptor de Morte Celular Programada 1 , Microambiente Tumoral
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