RESUMEN
Cancer vaccines are gaining ground as immunotherapy options. We have previously demonstrated in cutaneous melanoma (CM) patients that adjuvant treatment with VACCIMEL, a mixture of four irradiated CM cell lines co-adjuvanted with BCG and GM-CSF, increases the cellular immune response to melanocyte differentiation antigens, cancer-testis antigens and neoantigens, with respect to basal levels. On the other hand, it is also known that treatment with anti-PD-1 monoclonal antibodies (MAbs), acting on pre-existing tumor-reactive lymphocytes, induces clinical responses in CM patients, albeit in a fraction of treated patients. A combination of both treatments would appear therefore desirable. In this paper, we describe CM patients who, having progressed even years after vaccination, were treated with anti-PD-1 MAbs. In 5/5 of such progressor patients, complete responses were obtained which lasted between 3 and 65+ months. Three of the patients remain disease-free and two recurred. One of the patients passed away after a recurrence of brain metastases. We suggest that clonally expanded reactive lymphocytes induced by VACCIMEL partially remain as memory cells, which may be recalled after tumor recurrence and may foster ulterior activity of anti-PD-1 MAbs.
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Anticuerpos Monoclonales , Vacunas contra el Cáncer , Melanoma Cutáneo Maligno , Receptor de Muerte Celular Programada 1 , Neoplasias Cutáneas , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adyuvantes Inmunológicos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Melanoma Cutáneo Maligno/inmunología , Melanoma Cutáneo Maligno/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/terapia , Resultado del TratamientoRESUMEN
Introduction: Treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%. Methods: In this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months. Results: At the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (p<0.0001). Discussion: This NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Inhibidores de Proteínas Quinasas , Humanos , Células Asesinas Naturales , Inhibidores de Proteínas Quinasas/uso terapéutico , Inducción de RemisiónRESUMEN
Introduction: Tissue biomarkers that aid in identifying cutaneous melanoma (CM) patients who will benefit from adjuvant immunotherapy are of crucial interest. Metastatic tumor-draining lymph nodes (mTDLN) are the first encounter site between the metastatic CM cells and an organized immune structure. Therefore, their study may reveal mechanisms that could influence patients´ outcomes. Methods: Twenty-nine stage-III CM patients enrolled in clinical trials to study the vaccine VACCIMEL were included in this retrospective study. After radical mTDLN dissection, patients were treated with VACCIMEL (n=22) or IFNα-2b (n=6), unless rapid progression (n=1). Distant Metastasis-Free Survival (DMFS) was selected as an end-point. Two cohorts of patients were selected: one with a good outcome (GO) (n=17; median DMFS 130.0 months), and another with a bad outcome (BO) (n=12; median DMFS 8.5 months). We analyzed by immunohistochemistry and immunofluorescence the expression of relevant biomarkers to tumor-cell biology and immune cells and structures in mTDLN, both in the tumor and peritumoral areas. Results: In BO patients, highly replicating Ki-67+ tumor cells, low tumor HLA-I expression and abundant FoxP3+ lymphocytes were found (p=0.037; p=0.056 and p=0.021). In GO patients, the most favorable biomarkers for prolonged DMFS were the abundance of peri- and intra-tumoral CD11c+ cells (p=0.0002 and p=0.001), peri-tumoral DC-LAMP+ dendritic cells (DCs) (p=0.001), and PNAd+ High Endothelial Venules (HEVs) (p=0.004). Most strikingly, we describe in GO patients a peculiar, heterogeneous structure that we named FAPS (Favoring Antigen-Presenting Structure), a triad composed of DC, HEV and CD62L+ naïve lymphocytes, whose postulated role would be to favor tumor antigen (Ag) priming of incoming naïve lymphocytes. We also found in GO patients a preferential tumor infiltration of CD8+ and CD20+ lymphocytes (p=0.004 and p=0.027), as well as peritumoral CD20+ aggregates, with no CD21+ follicular dendritic cells detected (p=0.023). Heterogeneous infiltration with CD64+CD68-CD163-, CD64+CD68+CD163- and CD64+CD68+CD163+ macrophages were observed in both cohorts. Discussion: The analysis of mTDLN in GO and BO patients revealed marked differences. This work highlights the importance of analyzing resected mTDLN from CM patients and suggests a correlation between tumor and immune characteristics that may be associated with a spontaneous or vaccine-induced long DMFS. These results should be confirmed in prospective studies.
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Melanoma , Neoplasias Cutáneas , Humanos , Melanoma/terapia , Neoplasias Cutáneas/terapia , Vénulas , Estudios Prospectivos , Estudios Retrospectivos , Adyuvantes Inmunológicos , Adyuvantes Farmacéuticos , Ganglios Linfáticos , Inmunoterapia , Células Dendríticas , Melanoma Cutáneo MalignoRESUMEN
Intravesical Bacillus Calmette Guerin (BCG) is the gold standard therapy for intermediate/high-risk non-muscle invasive bladder cancer (NMIBC). However, the response rate is ~60%, and 50% of non-responders will progress to muscle-invasive disease. BCG induces massive local infiltration of inflammatory cells (Th1) and ultimately cytotoxic tumor elimination. We searched for predictive biomarker of BCG response by analyzing tumor-infiltrating lymphocyte (TIL) polarization in the tumor microenvironment (TME) in pre-treatment biopsies. Pre-treatment biopsies from patients with NMIBC who received adequate intravesical instillation of BCG (n = 32) were evaluated retrospectively by immunohistochemistry. TME polarization was assessed by quantifying the T-Bet+ (Th1) and GATA-3+ (Th2) lymphocyte ratio (G/T), and the density and degranulation of EPX+ eosinophils. In addition, PD-1/PD-L1 staining was quantified. The results correlated with BCG response. In most non-responders, Th1/Th2 markers were compared in pre-and post-BCG biopsies. ORR was 65.6% in the study population. BCG responders had a higher G/T ratio and a greater number of degranulated EPX+ cells. Variables combined into a Th2-score showed a significant association with higher scores in responders (p = 0.027). A Th2-score cut-off value >48.1 allowed discrimination of responders with 91% sensitivity but lower specificity. Relapse-free survival was significantly associated with the Th2-score (p = 0.007). In post-BCG biopsies from recurring patients, TILs increased Th2-polarization, probably reflecting BCG failure to induce a pro-inflammatory status and, thus, a lack of response. PD-L1/PD-1 expression was not associated with the response to BCG. Our results support the hypothesis that a pre-existing Th2-polarized TME predicts a better response to BCG, assuming a reversion to Th1 polarization and antitumor activity.
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Carcinoma , Neoplasias de la Vejiga Urinaria , Humanos , Estudios Retrospectivos , Vacuna BCG/uso terapéutico , Antígeno B7-H1 , Receptor de Muerte Celular Programada 1 , Microambiente Tumoral , Vejiga Urinaria , Recurrencia Local de Neoplasia , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , BiomarcadoresRESUMEN
Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ- and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ- or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C- NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ- and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C- NK cells retained greater functionality after treatment than FcεRIγ + NKG2C- NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.
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Neoplasias de la Mama , Infecciones por Citomegalovirus , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Citomegalovirus , Células Asesinas Naturales , Citocinas , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismoRESUMEN
Treatment-free remission (TFR) in chronic myeloid leukemia (CML) is safe under adequate molecular monitoring, but questions remain regarding which factors may be considered predictive for TFR. Argentina Stop Trial (AST) is a multicenter TFR trial showing that 65% of patients sustain molecular remission, and the prior time in deep molecular response (DMR) was associated with successful TFR. Luminex technology was used to characterize cytokines in plasma samples. Using machine learning algorithms, MCP-1 and IL-6 were identified as novel biomarkers and MCP-1low/IL-6low patients showed eightfold higher risk of relapse. These findings support the feasibility of TFR for patients in DMR and MCP-1/IL-6 plasma levels are strong predictive biomarkers.
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Interleucina-6 , Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Inhibidores de Proteínas Quinasas , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Biomarcadores , Inducción de Remisión , Resultado del TratamientoRESUMEN
BACKGROUND: Cutaneous melanoma is the skin cancer with the highest mutational burden and metastatic rate. Early genetic alterations and biomarkers of distant progression are a point of interest. In addition to germline-susceptibility loci, almost 30% of melanomas arise from precursor benign nevi lesions, providing a source for malignant transformation. CASE PRESENTATION: Patient#009 developed a cutaneous melanoma over a nevus, followed by progression to regional and distant metastases in months, unresponsive to targeted therapy. To search for the genetic contribution to this rapid progression, a longitudinal analysis was performed through WES of germline, nevi, primary tumor, and a metastatic lymph node. Differential SNP/INDEL and CNV gene alterations, with functional impact on key pathways and cancer hallmarks in each step of evolution, were discerned. Tumor-associated nevus was, for the first time, split into two sections, distant and adjacent to the primary tumor, to study its heterogeneity. Shared SNP alterations, with stable allele fraction from germline to metastasis were detected, mainly affecting DNA repair genes and promoting genome instability. Early somatic alterations, shared by nevi and primary and metastatic tumors, included BRAFV600E and focal copy-loss of several genes, acquiring additional cancer hallmarks. Phylogenetic analyses revealed that these common somatic alterations would provide a "bridge", allowing progression from a benign to a malignant state. Distant and adjacent nevi were rich in alterations, presenting differential SNP and CNV alterations. Upon tumor transformation, a marked increase in CNV over SNP alterations was determined. Both the number of SNP and CNV-affected genes, including known driver genes, increased throughout progression, although TMB levels remained lower than expected for melanoma. Typical alterations in BRAFV600E tumors related to intrinsic resistance to targeted therapy were found, including BRAF amplification and loss of PTEN, CDKN2A/B, and TP53 surveillance genes. Finally, numerous metastatic alterations were detected, further promoting tumor progression. CONCLUSIONS: In this patient, longitudinal WES analysis revealed a sequential and cumulative pattern of genetic alterations, where germline and nevi somatic events contributed early to its rapid clinical progression. In this case report, we found tumor-associated nevi as genetically heterogeneous precursor entities, in which potential prognostic biomarkers should be studied prospectively.
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Melanoma , Nevo , Neoplasias Cutáneas , Humanos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Melanoma/genética , Melanoma/patología , Proteínas Proto-Oncogénicas B-raf/genética , Filogenia , Transformación Celular Neoplásica , Melanoma Cutáneo MalignoRESUMEN
The CSF-470 vaccine (VACCIMEL) plus BCG and GM-CSF as adjuvants has been assayed in cutaneous melanoma patients. In the adjuvant randomized Phase II study CASVAC-0401, vaccinated patients had longer distant metastasis-free survival (DMFS) than those treated with IFNα2b. Five years after locking the data, an actualization was performed. The benefit in DMFS was maintained in the vaccinated group versus the IFNα2b-treated group (p = 0.035), with a median DMFS of 96 months for VACCIMEL and 13 months for IFNα2b. The favorable risk-benefit ratio was maintained. DMFS was also analyzed as a single cohort in all the IIB, IIC, and III patients (n = 30) who had been treated with VACCIMEL. The median DMFS was 169 months, and at 48 months follow-up, it was 71.4%, which was not statistically different from DMFS of previously published results obtained in adjuvancy with ipilimumab, pembrolizumab, nivolumab, or dabrafenib/trametinib. The possible toxicity of combining VACCIMEL with anti-immune checkpoint inhibitors (ICKi) was analyzed, especially since VACCIMEL was co-adjuvated with BCG in every vaccination. A patient with in-transit metastases was studied to produce a proof of concept. During treatment with VACCIMEL, the patient developed T-cell clones reactive towards tumor-associated antigens. Three years after ending the VACCIMEL study, the patient progressed and was treated with ICKi. During ICKi treatment, the patient did not reveal any toxicity due to previous BCG treatment. When she recurred after a 4-year treatment with nivolumab, a biopsy was obtained and immunohistochemistry and RNA-seq were performed. The tumor maintained expression of tumor-associated antigens and HLA-I and immune infiltration, with immunoreactive and immunosuppressive features. VACCIMEL plus BCG and GM-CSF is an effective treatment in adjuvancy for stages IIB, IIC, and III cutaneous melanoma patients, and it is compatible with subsequent treatments with ICKi.
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Vacunas contra el Cáncer , Trasplante de Células Madre Hematopoyéticas , Melanoma , Neoplasias Cutáneas , Adyuvantes Inmunológicos , Antígenos de Neoplasias , Vacuna BCG , Vacunas contra el Cáncer/efectos adversos , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Nivolumab/uso terapéutico , Melanoma Cutáneo MalignoRESUMEN
Triple Negative Breast Cancer (TNBC) treatment is still challenging, and immunotherapy is a potential approach in this tumor subtype. Cetuximab is an IgG1 monoclonal antibody (mAb) directed against Epidermic Growth Factor Receptor (EGFR), a protein overexpressed in a subgroup of TNBC patients and associated with poor prognosis. Previously, we demonstrated in vitro that Cetuximab triggers Ab-dependent cell cytotoxicity against TNBC cells. In this study, using co-cultures including TNBC cells, and NK and Dendritic Cells (DCs) from healthy donors, we studied the effect of Cetuximab-activated NK cells on DC function. Given that we already demonstrated that TNBC has an immunosuppressive effect on NK cells, we also tested Cetuximab combination with IL-15. We determined that Cetuximab opsonization of TNBC cells increased IFN-γ and TNF-α production by NK cells co-cultured with DCs. Moreover, we showed that NK cells activated by TNBC cells opsonized with Cetuximab promoted tumor material uptake and maturation of DCs, as well as their ability to produce IL-12. Furthermore, the stimulation with IL-15 increased the activation of NK cells and the maturation of DCs. These results suggest that IL-15 may enhance the efficacy of Cetuximab in the treatment of TNBC by promoting activation of both NK cells and DCs.
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Cetuximab/farmacología , Interleucina-15/farmacología , Línea Celular Tumoral , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Femenino , Citometría de Flujo , Humanos , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Fagocitosis/efectos de los fármacos , Neoplasias de la Mama Triple Negativas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The CSF-470 vaccine consists of lethally-irradiated allogeneic cells derived from four cutaneous melanoma cell lines administered plus BCG and GM-CSF as adjuvants. In an adjuvant phase II study vs. IFN-α2b, the vaccine significantly prolonged the distant metastasis-free survival (DMFS) of stages IIB-IIC-III melanoma patients with evidence of the induction of immune responses against vaccine cells. Purpose: The aim of this study was to analyze the antigens against which the immune response was induced, as well as the T-helper profile and lytic ability of immune cells after CSF-470 treatment. Methods: HLA-restricted peptides from tumor-associated antigens (TAAs) were selected from TANTIGEN database for 13 evaluable vaccinated patients. In addition, for patient #006 (pt#006), tumor somatic variants were identified by NGS and candidate neoAgs were selected by predicted HLA binding affinity and similarity between wild type (wt) and mutant peptides. The patient's PBMC reactivity against selected peptides was detected by IFNγ-ELISPOT. T-helper transcriptional profile was determined by quantifying GATA-3, T-bet, and FOXP3 mRNA by RT-PCR, and intracellular cytokines were analyzed by flow cytometry. Autologous tumor cell lysis by PBMC was assessed in an in vitro calcein release assay. Results: Vaccinated patient's PBMC reactivity against selected TAAs derived peptides showed a progressive increase in the number of IFNγ-producing cells throughout the 2-yr vaccination protocol. ELISPOT response correlated with delayed type hypersensitivity (DTH) reaction to CSF-470 vaccine cells. Early upregulation of GATA-3 and Foxp3 mRNA, as well as an increase in CD4+IL4+cells, was associated with a low DMFS. Also, IFNγ response against 9/73 predicted neoAgs was evidenced in the case of pt#006; 7/9 emerged after vaccination. We verified in pt# 006 that post-vaccination PBMC boosted in vitro with the vaccine lysate were able to lyse autologous tumor cells. Conclusions: A progressive increase in the immune response against TAAs expressed in the vaccine and in the patient's tumor was induced by CSF-470 vaccination. In pt#006, we demonstrated immune recognition of patient's specific neoAgs, which emerged after vaccination. These results suggest that an initial response against shared TAAs could further stimulate an immune response against autologous tumor neoAgs.
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Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Melanoma/inmunología , Melanoma/terapia , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/terapia , Linfocitos T/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Células Alogénicas , Vacuna BCG/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Humanos , Inmunoterapia Adoptiva/métodos , Melanoma Cutáneo MalignoRESUMEN
Objectives The quantitation of BCR-ABL1 mRNA is mandatory for chronic myeloid leukemia (CML) patients, and RT-qPCR is the most extensively used method in testing laboratories worldwide. Nevertheless, substantial variation in RT-qPCR results makes inter-laboratory comparability hard. To facilitate inter-laboratory comparative assessment, an international scale (IS) for BCR-ABL1 was proposed. Methods The laboratory-specific conversion factor (CF) to the IS can be derived from the World Health Organization (WHO) genetic reference panel; however, this material is limited to the manufacturers to produce and calibrate secondary reference reagents. Therefore, we developed secondary reference calibrators, as lyophilized cellular material, aligned to the IS. Our purpose was both to re-evaluate the CF in 18 previously harmonized laboratories and to propagate the IS to new laboratories. Results Our field trial including 30 laboratories across Latin America showed that, after correction of raw BCR-ABL1/ABL1 ratios using CF, the relative mean bias was significantly reduced. We also performed a follow-up of participating laboratories by annually revalidating the process; our results support the need for continuous revalidation of CFs. All participating laboratories also received a calibrator to determine the limit of quantification (LOQ); 90% of them could reproducibly detect BCR-ABL1, indicating that these laboratories can report a consistent deep molecular response. In addition, aiming to investigate the variability of BCR-ABL1 measurements across different RNA inputs, we calculated PCR efficiency for each individual assay by using different amounts of RNA. Conclusions In conclusion, for the first time in Latin America, we have successfully organized a harmonization platform for BCR-ABL1 measurement that could be of immediate clinical benefit for monitoring the molecular response of patients in low-resource regions.
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Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Calibración , Humanos , América Latina , Control de Calidad , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
It is estimated that ~50% of patients with melanoma harbour BRaf (BRAF)V600 driver mutations, with the most common of these being BRAFV600E, which leads to the activation of mitogenactivated protein kinase proliferative and survival pathways. BRAF inhibitors are used extensively to treat BRAFmutated metastatic melanoma; however, acquired resistance occurs in the majority of patients. The effects of longterm treatment with PLX4032 (BRAFV600 inhibitor) were studied in vitro on sensitive V600E BRAFmutated melanoma cell lines. After several weeks of treatment with PLX4032, the majority of the melanoma cells died; however, a proportion of cells remained viable and quiescent, presenting senescent cancer stem celllike characteristics. This surviving population was termed SUR cells, as discontinuing treatment allowed the population to regrow while retaining equal drug sensitivity to that of parental cells. RNA sequencing analysis revealed that SUR cells exhibit changes in the expression of 1,415 genes (P<0.05) compared with parental cells. Changes in the expression levels of a number of epigenetic regulators were also observed. These changes and the reversible nature of the senescence state were consistent with epigenetic regulation; thus, it was investigated as to whether the senescent state could be reversed by epigenetic inhibitors. It was found that both parental and SUR cells were sensitive to different histone deacetylase (HDAC) inhibitors, such as SAHA and MGCD0103, and to the cyclindependent kinase (CDK)9 inhibitor, CDKI73, which induced apoptosis and reduced proliferation both in the parental and SUR populations. The results suggested that the combination of PLX4032 with HDAC and CDK9 inhibitors may achieve complete elimination of SUR cells that persist after BRAF inhibitor treatment, and reduce the development of resistance to BRAF inhibitors.
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Redes Reguladoras de Genes/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/farmacología , Sulfonamidas/farmacología , Vemurafenib/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Melanoma/tratamiento farmacológico , Mutación , Análisis de Secuencia de ARNRESUMEN
Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of LSC-enriched CD34+CD38-CD26+ and normal hematopoietic stem cells (HSC) fractions obtained from the same chronic phase (CP) CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC-enriched CD34+CD38-CD26+ and HSC fractions from CML-CP patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML-CP patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. We confirmed by RT-qPCR that the levels of miR-196a-5p were increased more than nine-fold in CD26+ (BCR-ABL1 + ) vs. CD26- (BCR-ABL1 -) CD34+CD38- fractions from CML-CP patients at diagnosis, and in silico analysis revealed a significant association to lipid metabolism and hematopoiesis functions. In the light of recent descriptions of increased oxidative metabolism in CML LSC-enriched fractions, these results serve as a guide for future functional studies that evaluate the role of microRNAs in this process. Metabolic vulnerabilities in LSCs open the road for new therapeutic strategies. This is the first report of the miRNome of CML-CP CD34+CD38- fractions that distinguishes between CD26+ (BCR-ABL1 + ) and their CD26- (BCR-ABL1 - ) counterparts, providing valuable data for future studies.
RESUMEN
The CSF-470 cellular vaccine plus BCG and rhGM-CSF increased distant metastases-free survival in cutaneous melanoma patients stages IIB-IIC-III relative to medium dose IFN-α2b (CASVAC-0401 study). Patient-045 developed a mature vaccination site (VAC-SITE) and a regional cutaneous metastasis (C-MTS), which were excised during the protocol, remaining disease-free 36 months from vaccination start. CDR3-TCRß repertoire sequencing in PBMC and tissue samples, along with skin-DTH score and IFN-γ ELISPOT assay, were performed to analyze the T-cell immune response dynamics throughout the immunization protocol. Histopathological analysis of the VAC-SITE revealed a highly-inflamed granulomatous structure encircled by CD11c+ nested-clusters, brisk CD8+ and scarce FOXP3+, lymphocytes with numerous Langhans multinucleated-giant-cells and macrophages. A large tumor-regression area fulfilled the C-MTS with brisk lymphocyte infiltration, mainly composed of CD8+PD1+ T-cells, CD20+ B-cells, and scarce FOXP3+ cells. Increasing DTH score and IFN-γ ELISPOT assay signal against the CSF-470 vaccine-lysate was evidenced throughout immunization. TCRß repertoire analysis revealed for the first time the presence of common clonotypes between a VAC-SITE and a C-MTS; most of them persisted in blood by the end of the immunization protocol. In vitro boost with vaccine-lysate revealed the expansion of persistent clones that infiltrated the VAC-SITE and/or the C-MTS; other persistent clones expanded in the patient's blood as well. We propose that expansion of such persistent clonotypes might derive from two different although complementary mechanisms: the proliferation of specific clones as well as the expansion of redundant clones, which increased the number of nucleotide rearrangements per clonotype, suggesting a functional antigenic selection. In this patient, immunization with the CSF-470 vaccine plus BCG and rhGM-CSF induced a T-cell repertoire at the VAC-SITE that was able to infiltrate an emerging C-MTS, which resulted in the expansion of a T-cell repertoire that persisted in blood by the end of the 2-year treatment.
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Vacunas contra el Cáncer/uso terapéutico , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/inmunología , Vacuna BCG/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Melanoma Cutáneo MalignoRESUMEN
Patients with non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) have shown benefit from anti-PD-1 therapies. However, not all patients experience tumor shrinkage, durable responses or prolonged survival, demonstrating the need to find response markers. In blood samples from NSCLC and RCC patients obtained before and after anti-PD-1 treatment, we studied leukocytes by complete blood cell count, lymphocyte subsets using flow cytometry and plasma concentration of nine soluble mediators, in order to find predictive biomarkers of response and to study changes produced after anti-PD-1 therapy. In baseline samples, discriminant analysis revealed a combination of four variables that helped differentiate stable disease-response (SD-R) from progressive disease (PD) patients: augmented frequency of central memory CD4+ T cells and leukocyte count was associated with response while increased percentage of PD-L1+ natural killer cells and naïve CD4+ T cells was associated with lack of response. After therapy, differential changes between responders and non-responders were found in leukocytes, T cells and TIM-3+ T cells. Patients with progressive disease showed an increase in the frequency of TIM-3 expressing CD4+ and CD8+ T cells, whereas SD-R patients showed a decrease in these subsets. Our findings indicate that a combination of immune variables from peripheral blood (PB) could be useful to distinguish response groups in NSCLC and RCC patients treated with anti-PD-1 therapy. Frequency of TIM-3+ T cells showed differential changes after treatment in PD vs SD-R patients, suggesting that it may be an interesting marker for monitoring progression during therapy.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/inmunología , Anciano , Proteína C-Reactiva/análisis , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Células Renales/inmunología , Femenino , Receptor 2 Celular del Virus de la Hepatitis A/sangre , Humanos , Neoplasias Renales/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/inmunología , Masculino , Persona de Mediana EdadRESUMEN
A major obstacle to obtaining relevant results in cancer vaccination has been the lack of identification of immunogenic antigens. Dendritic cell (DC)-based cancer vaccines used preventively may afford protection against tumor inoculation, but the effect of antigen choice on anti-tumor protection is not clear. When using irradiated syngeneic tumor cells to load DCs, tumor self-antigens are provided, including tumor-associated antigens (TAAs) and neoantigens generated by tumor mutations. On the other hand, allogeneic tumor cells could only supply shared TAAs. To assess the advantages of each source in protective vaccination, we analyzed in C57BL/6 mice the effect of loading DCs with irradiated syngeneic B16-F1 or allogeneic Cloudman melanoma cells; both cell lines were characterized by whole exome sequencing and RNAseq. Tumor cell components from the two irradiated cell lines were efficiently internalized by DCs, and transported to MHC-class II positive tubulovesicular compartments (MIICs). DCs loaded with allogeneic irradiated Cloudman cells (DC-ApoNecALLO) induced a partially effective anti-melanoma protection, although Cloudman and B16-F1 cells share the expression of melanocyte differentiation antigens (MDAs), cancer-testis antigens (CTAs) and other TAAs. DCs loaded with syngeneic B16-F1 cells (DC-ApoNecSYN) established a more potent and long-lasting protection and induced a humoral anti-B16F1 response, thus suggesting that neoepitopes are needed for inducing long-lasting protection.
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Células Alogénicas/inmunología , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Melanoma Experimental/inmunología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Inmunidad Humoral , Masculino , Melanoma Experimental/prevención & control , Ratones , Ratones Endogámicos C57BL , Organismos Libres de Patógenos Específicos , VacunaciónRESUMEN
As cutaneous melanoma (CM) currently remains with a bleak prognosis, thorough investigation of new treatment options are of utmost relevance. In the phase II/III randomized clinical trial (CASVAC-0401), the repeated immunization of stages IIB-III CM patients with the irradiated, allogeneic cellular CSF-470 vaccine plus the adjuvants bacillus Calmette-Guerin (BCG) and recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) demonstrated a significant benefit over IFN-alpha2B treatment in distant metastasis-free survival. Here we present on the short and long term immune monitoring results after completing the 2-year protocol; a continuation of the previous report by Mordoh et al. (1). We demonstrate that the repeated CSF-470 vaccinations stimulated a long term cellular and humoral immunity response directed against the vaccine antigens. In the case of 2 patients, we are able to show that a similar immune response was generated against autologous antigens. Evaluation of inhibitory receptor co-expression on patient's T cells indicates that the vaccination protocol did not stimulate T cell exhaustion. In order to better understand the basis for the efficacious vaccine responses observed, we investigated the short term immune events following vaccine injection. A significant increase in C-reactive protein (CRP) and IL-6 was observed 24 h after vaccination, with in vitro studies suggesting IL-6 production occurs in the vaccine site. We demonstrate that CRP enhances the cytotoxicity of peripheral blood mononuclear cells (PBMC) against melanoma cells in an in vitro model. Additionally, CRP stimulates the release of pro and anti-inflammatory cytokines from PBMC. As our results demonstrate that successive vaccinations with CSF-470 plus adjuvants promoted an increase in both anti-tumor innate and adaptive immunity, we propose a subsequent model of action.
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Proteína C-Reactiva/metabolismo , Vacunas contra el Cáncer/inmunología , Interleucina-6/metabolismo , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Linfocitos T/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Antígenos de Neoplasias/inmunología , Autoantígenos/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Estudios de Seguimiento , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunización , Mediadores de Inflamación/metabolismo , Interferón gamma/metabolismo , Modelos Inmunológicos , Estadificación de Neoplasias , Ensayos Clínicos Controlados Aleatorios como Asunto , VacunaciónRESUMEN
The standard treatment for Triple Negative Breast Cancer (TNBC) patients is cytotoxic chemotherapy, but it is restricted since the duration of response is usually short. Blocking the PD-1/PD-L1 pathway through monoclonal antibodies (mAbs) appears to be a promising therapeutic strategy for TNBC patients. Avelumab is a human IgG1 anti-PD-L1 mAb being tested in clinical trials that may also trigger antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells as an additional antitumor activity. In the present work, we studied in vitro Avelumab-mediated ADCC against a panel of TNBC cells with different PD-L1 expression using peripheral blood mononuclear cells (PBMC) or purified NK cells from healthy donors. We determined that Avelumab significantly enhanced NK-cell mediated cytotoxicity against TNBC cells and that tumor cells expressing higher levels of PD-L1 were more sensitive to Avelumab-mediated ADCC. IFN-γ treatment upregulated PD-L1 expression in tumor cells but had a variable impact on Avelumab-mediated ADCC, which could be related to the simultaneous effect of IFN-γ on the expression of NK cell ligands. Moreover, IL-2 and IL-15 stimulation of NK cells enhanced Avelumab-triggered cytokine production and degranulation along with increased lytic activity against tumor cells. Improving the treatment of TNBC remains still a considerable challenge. This in vitro study suggests that Avelumab-mediated ADCC, independently of the blockade of the PD-1/PD-L1 pathway, could be a valuable mechanism for tumor cell elimination in TNBC. Avelumab combination with immunomodulators such as IL-15 or IL-2 could be taken into consideration to increase the therapeutic efficacy of Avelumab in TNBC.
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Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antineoplásicos Inmunológicos/farmacología , Células Asesinas Naturales/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Quantification of BCR-ABL1 mRNA levels in peripheral blood of chronic myeloid leukemia patients is a strong indicator of response to tyrosine-kinase inhibitors (TKI) treatment. However, additional prognostic markers are needed in order to better classify patients. The hypothesis of leukemic stem cells (LSCs) heterogeneity and persistence, suggests that their functional evaluation could be of clinical interest. In this work, we assessed the primitive and progenitor fractions in patients at diagnosis and during TKI treatment using functional in vitro assays, defining a "functional leukemic burden" (FLB). We observed that the FLB was reduced in vivo in both fractions upon treatment. However, different FLB levels were observed among patients according to their response to treatment, suggesting that quantification of the FLB could complement early molecular monitoring. Given that FLB assessment is limited by BCR-ABL1 mRNA expression levels, we developed a novel detection method of primitive cells at the DNA level, using patient-specific primers and direct nested PCR in colonies obtained from functional in vitro assays. We believe that this method could be useful in the context of discontinuation trials, given that it is unknown whether the persistent leukemic clone represents LSCs, able to resume the leukemia upon TKI removal.
RESUMEN
The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFNα2b in the distant metastasis-free survival for stages IIB-IIC-III cutaneous melanoma patients in a randomized phase II/III clinical trial (CASVAC-0401, NCT01729663). At the end of the 2-year CSF-470 immunization protocol, patient #006 developed several lung and one subcutaneous melanoma metastases; this later was excised. In this report, we analyzed the changes throughout vaccination of immune populations in blood and in the tumor tissue, with special focus on the T-cell repertoire. Immunohistochemistry revealed a marked increase in CD8+, CD4+, and CD20+ lymphocytes infiltrating the metastasis relative to the primary tumor. Lymphocytes were firmly attached to dying-tumor cells containing Granzyme-B granules. Whole-exon sequencing assessment indicated a moderate-to-high tumor mutational burden, with BRAFV600E as the main oncogenic driver. Mutational signature presented large numbers of mutations at dipyrimidines, typical of melanoma. Relevant tumor and immune-related genes from the subcutaneous metastasis were addressed by RNA-Seq analysis, revealing expression of typical melanoma antigens and proliferative tumor-related genes. Stimulatory and inhibitory immune transcripts were detected as well as evidence of active T-cell effector function. Peripheral blood monitoring revealed an increase in CD4+ and CD8+ cells by the end of the immunization protocol. By CDR3-T-cell receptor ß (TCRß) sequencing, generation of new clones and an increase in oligoclonality was observed in the peripheral T-cells immune repertoire throughout immunization. A shift, with the expansion of selected preexisting and newly arising clones with reduction of others, was detected in blood. In tumor-infiltrating lymphocytes, prevalent clones (50%) were both new and preexisting that were expanded in blood following CSF-470 immunization. These clones persisted in time, since 2 years after completing the immunization, 51% of the clones present in the metastasis were still detected in blood. This is the first report of the modulation of the TCRß repertoire from a melanoma patient immunized with the CSF-470 vaccine. After immunization, the changes observed in peripheral immune populations as well as in the tumor compartment suggest that the vaccine can induce an antitumor adaptive immune repertoire that can reach tumor lesions and persists in blood for at least 2 years.