Tumor infiltrating lymphocytes (TILs) are known to be a prognostic and predictive biomarker in breast cancer, particularly in triple negative breast cancer (TNBC) patients. International guidelines ...have been proposed to evaluate them in the clinical setting as a continuous variable, without a clear defined cut-off. However, there are scenarios where the immune infiltration is heterogeneous that some areas of the patient's tumour have high numbers of TILs while other areas completely lack them. This spontaneous presentation of a heterogeneous immune infiltration could be a great opportunity to study why some tumours present TILs at diagnosis but others do not, while eliminating inter patient's differences.
In this study, we have identified five TNBC patients that showed great TIL heterogeneity, with areas of low (≤5%) and high (≥50%) numbers of TILs in their surgical specimens. To evaluate immune infiltration heterogeneity, we performed and analyzed bulk RNA-sequencing in three independent triplicates from the high and low TIL areas of each patient.
Gene expression was homogeneous within the triplicates in each area but was remarkable different between TILs regions. These differences were not only due to the presence of TILs as there were other non-inflammatory genes and pathways differentially expressed between the two areas.
This highlights the importance of intratumour heterogeneity driving the immune infiltration, and not patient's characteristics like the HLA phenotype, germline DNA or immune repertoire.
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•EM1 and UVB1 reduce the viability of cells derived from HER2-positive and TNBC PDXs.•UVB1 has antiproliferative actions in Trastuzumab-emtansine (T-DM1) resistant cells.•UVB1 impairs ...the organoids formation capacity in T-DM1 resistant cells.•UVB1 inhibits the growth of established organoids derived from T-DM1 resistant cells.•VDR expression in PDXs positively correlates with UVB1 antitumor response.
Despite advances in breast cancer (BC) treatment, its mortality remains high due to intrinsic or acquired resistance to therapy. Several ongoing efforts are being made to develop novel drugs to treat this pathology with the aim to overcome resistance, prolong patient survival and improve their quality of life. We have previously shown that the non-hypercalcemic vitamin D analogues EM1 and UVB1 display antitumor effects in preclinical studies employing conventional cell lines and animal models developed from these cells. In this work, we explored the antitumor effects of EM1 and UVB1 employing BC cells derived from patient-derived xenografts (PDXs), which are a powerful preclinical tool for testing new drugs. We demonstrated that the analogues reduced the viability of HER2-positive and Triple Negative BC-PDXs. Moreover, using an in vitro model of acquired resistance to Trastuzumab-emtansine, UVB1 displayed anti-proliferative actions under 2D and 3D culture conditions. It inhibited both formation and growth of established organoids. In addition, a direct correlation between UVB1 antitumor effects and VDR expression in PDXs was found. In conclusion, all the results reinforce the potential use of these vitamin D analogues as antitumor agents to treat HER2-positive and Triple Negative BC.
To assess the ability of urgent head computed tomography (CT) scan screening to detect patients who can evolve to brain death (BD).
Patients who underwent urgent head CT scan and meet the following ...criteria: midline shift greater than 5mm and/or decrease or absence of basal cisterns. A follow-up for 28 days of each patient was made. Epidemiological data (sex, age, cause of brain injury), clinical data (level of consciousness, severity index in the CT) and patient outcomes (death, BD, discharge or transfer) were recorded. This was a prospective observational study.
One hundred and sixty-six patients were selected for study, with mean age 60.08 (SD 21.8) years. A percentage of 49.4 were men and the rest women. In the follow-up, 20,5% (n=34) had BD. In univariate analysis, intracerebral hemorrhage, Glasgow Coma Scale score less than 8 and alteration of basal cisterns were statistically significant in predicting BD (P<.05). Multivariate analysis showed that patients with compression of basal cisterns were 20 (95% confidence interval 95% CI 2.61 to 153.78; P=.004 times more likely to progress to brain death, while the absence there of 62.6 (95% CI 13.1 to 738.8; P<.001 times more.
Our work shows that data as easy to interpret as compression/absence of basal cisterns can be a powerful tool for screening patients at risk for progression to BD.
Antibody-drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is ...approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as vic-trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression.
Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.
Abstract
In the recent times the way to tackle cancer has been broadly expanded to new opportunities, being the immunotherapy the spearhead of this revolution. Two of the different novel strategies ...to boost the immune response against tumor cells are the T Cell Bispecific antibodies (TCBs) and the Chimeric Antigen Receptors (CARs), both able to redirect the T cells of the host against the tumor cells. While the TCBs consist of an engineered antibody that binds both to a tumor associated antigen and a T cell receptor, activating it; the CARs consist of a modified T cell that exhibit the binding site to the antigen fused to the intracellular signaling motifs that activate the T cell. Clinical evidence shows that either TCBs or CARs are effective to treat cancer. These can be directed to a vast variety of tumor antigens. Some examples are the HER2-TCB, directed against the HER2 receptor tyrosine kinase overexpressed in 20% of the breast cancers. However, the therapeutic effectivity in tumors is lower than expected and the mechanisms of resistance to these promising immunotherapies have not been explored yet. Therefore, there is a need to anticipate them. In this project, we have developed a immunoresistant model against HER2-TCB, by using the HER2+ breast cancer cell line BT474 as a model. To generate immunoresistant cells, cells were exposed with the TCBs and peripheral blood mononuclear cells (PBMCs) for several months in order to obtain resistance. Indeed, these resistant cells were resistant also in a 3D organoid model and in an in vivo humanized PBMC model. Furthermore, these resistant cells were also resistant to a CAR expressing HER2. Interestingly, a cytokine array that assess more than 80 cytokines did not show a dramatic change in the secretome of resistant cells, in basal conditions and in presence of PBMCs and HER2-TCB, suggesting that the mechanism of resistant is intrinsic on the resistant cells. In addition, these resistant cells did not lose the expression of the antigen (HER2). Therefore, we aimed to identify intrinsic mechanisms of resistance by means of RNA-seq. Although we did not identify the cause of the resistance yet, transcriptomic analysis showed a dramatic change between sensitive and resistant cells, with more than 50 genes acutely up-or downregulated in resistant cells (≥4-fold; p < 10-5). Gene set enrichment analysis identified numerous differences in biological processes between both populations. In parallel, we have also generated the very same model in vivo, with serial passagues in humanized PBMC mice treated with HER2-TCB. The next step will be to identify the mechanism of resistance by gain and loss of function approaches and to validate it in a cohort of human samples and in our collection of Patient Derived Xenografts. In conclusion, we have generated an interesting resistant model to immunotherapies that can be used as a tool to identify unknown mechanisms of resistance and with potential clinical applications that will reinforce the use of immunotherapy in the clinics.
Citation Format: Enrique Javier Arenas Lahuerta, Alex Martínez-Sabadell, Irene Rius Ruiz, Beatriz Morancho, Macarena Román, Cristina Bernadó, Joaquín Arribas. Identification of novel mechanisms of resistance against the redirection of T-cell effector function for cancer therapy abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5570.
Abstract
Introduction: Elimination of cancer cells by effector immune cells represents the culmination of a complex cascade of events, and disruption of any of those events may result in resistance. ...T cell-engaging therapies, such as T cell bispecific antibodies (TCBs) or chimeric antigen receptors (CARs), are raising extraordinary expectations as future treatments for virtually all cancers. Encouraging these expectations, TCBs and CARs have been recently approved to treat some hematologic malignancies. In contrast, TCBs and CARs against solid tumors tested to date, have failed to show clinical efficacy. This failure prompted intense research and the subsequent identification of mechanisms of primary and acquired resistance. Different strategies are being implemented to overcome these mechanisms of resistance. All these mechanisms impinge on the ability of T cells to reach cancer cells and/or on the inhibition of T cells. However, little is known about putative intrinsic mechanisms of resistance of cancer cells. That is, mechanisms deployed by tumor cells to resist killing by fully active and correctly engaged T cells. In this study, we attempted to identify novel intrinsic mechanisms of resistance.
Methods: We have used TCBs and CARs targeting the cell surface receptor HER2 to identify a widespread mechanism of resistance to redirected T cells, using HER2-driven cell lines and Patient-Derived Xenografts (PDX).
Results: We have generated a model of intrinsic resistance to a TCB targeting HER2 by treating during 6 months co-cultures of PBMCs and parental BT474 cells. These resistant cells, named as BT-R, are also resistant to a HER2-CAR, in vitro and in vivo. Using this model, we identified by RNA-seq, a downmodulation of the IFN-gamma signaling pathway. Interestingly, using gain and loss of function approaches, we demonstrated that JAK2 loss is the cause of IFN-gamma deficient response, and as a consequence, resistance to HER2-TCB and CAR-HER2.
Conclusion: We have identified that the kinase JAK2, which transduces the signal initiated by interferon-gamma, is the component preferably disrupted to acquired resistance in all resistant models developed in vitro and in vivo. These results unveil a novel mechanism of resistance to T-cell based therapies, and imply the potential use of JAK2 and IFN-gamma response as a surrogate biomarker of response to immunotherapies. In addition, they open the avenue for the screening for therapies that can overcome deficient interferon-gamma response or restore JAK2 levels, which are promising potential candidates to increase the benefits of immunotherapies.
Citation Format: Enrique Javier Arenas Lahuerta, Alex Martínez-Sabadell, Irene Rius Ruiz, Macarena Román Alonso, Marta Escorihuela, Antonio Luque, Carlos A. Fajardo, Alena Gros, Christian Klein, Joaquín Arribas. JAK2 downmodulation leads to interferon gamma deficient response and resistance to immunotherapy in breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1690.
Day by day, the rational use of water is becoming more important in fruit production. The aim of this study was to evaluate the effect of using double drip line in pear (Pyrus communis) production, ...cv. Triunfo de Viena in comparison with the traditional system of one drip line per plant row. The research was based on the application of two specific processes, one consisting of a drip line per plant row with six emitters of 8 lt h-1 and another with two drip lines per plant row with three emitters of 8 lt h.sup.-1 each. Watermark sensors were used to measure and control soil matric potential. It covered 100% of potential evapotranspiration, which was 55.5 mm month-1. The results of the variables allow ground water irrigation scheduling and ensure adequate water supply during the period of water deficit. The double-line treatment did not differ significantly from the control in terms of production and fruit quality, which indicates that to use only one drip line will allow to get a similar yield and quality at a lower initial equipment cost. Key words: Production, Pyrus communis, quality, trickle irrigation. El objetivo del presente estudio fue evaluar el efecto de la utilización del sistema de doble línea de riego vs. el tradicional de una línea por hilera de plantas en la producción y calidad de la pera (Pyrus communis) variedad Triunfo de Viena. La investigación se basó en la aplicación de dos tratamientos definidos, uno consistente en una línea de goteo por hilera de plantas con seis emisores de 8 lt/h y otro, con dos líneas de goteo por hilera de plantas con tres emisores de 8 lt/h cada uno Se utilizaron sensores Watermark para medir y controlar el potencial matricial del suelo. Se cubrió el 100% de la evapotranspiración potencial que fue de 55.5 mm/mes. Los resultados obtenidos de las variables hídricas del suelo permitieron programar el riego y garantizar el suministro de agua adecuado en el periodo de déficit hídrico. El tratamiento de doble línea no difirió significativamente del control, lo que indica que utilizar una línea de riego permite obtener una producción y calidad similares con un menor costo inicial del equipo. Palabras clave: Calidad, producción de frutos, Pyrus communis, riego por goteo.
This study presents the analysis of the variables that have the greatest impact on energy requirements for an artificial lift system applied to extra heavy crude oils, considering an uncertainty ...behavior analysis through their sensitivity in the vertical flow modeling implemented for a Chichimene Field well. The selected variables are the viscosity and fluid density, the required artificial lift system pressure differential, well depth, the flow rate of produced fluids and the dilution percentage. The oil produced in this field has a density of 7,8 API, and the well studied features a water cut of about 10% and produces a total of 2400 BOD. For this flow naphtha dilution rates were defined using up to 20% by volume. The ranges of energy required for the lifting system for different scenarios raised by the analysis variables were also determined. For these conditions a variation of the energy required 20% for a fluid flow incremental of 50 BFOD was obtained, as established from the flow capacity of the well and the pressure required for sustaining a pressure head of 100 psi and 400 psi. Bottom dilution scheme establishes a change in artificial lift system energy requirement, of up to 25% for a 15% of diluter, whereas the relationship between the volumes produced and the system arrays gives an energy efficiency of 40%.
This paper presents the coverage measurements and path loss results of a 5G indoor and outdoor signal within the 5G Laboratory located in the Department of Electrical Engineering of the University of ...Chile. An Ericsson AIR 5121 antenna and commercial base station that is in a fixed position inside the laboratory and a 5G router equipment (5G NR mmWave Mobile Hotspot) are used to perform measurements in the 28 GHz frequency band. Both Line of Sight (LoS) indoor and Non-Line of Sight (NLoS) scenarios were considered within the laboratory and in a hall next to the it and Line of Sight (LoS) outdoor the laboratory. The measurement results show acceptable data rates in DL and UL, however a deviation of 20 dB to 30 dB with respect to the 3GPP TR 38.901, METIS and NYU model. These results are expected to be important to review the coverage behavior of a commercial 5G network at the 28 GHz band millimeter wave frequency.
Abstract
Introduction HER2+ correlates with aggressive phenotype and poor prognosis. The use of several antiHER2 drugs has dramatically improved prognosis and survival of HER2+ breast cancer (BC) ...patients. However, ≈20% of these patients will present resistance to antiHER2 therapies and relapse. The purpose of this study is to explore the role of AXL in antiHER2 treatment resistance and its potential as a druggable biomarker in HER2+ BC.
Methods We generated 2 trastuzumab (T) resistant models from a HER2+ BC patient. The first one was a PDX derived resistant cell line established by chronic treatment for 6 months with T in vitro (TR1 and TR2). The second resistant model (TR4) was established by chronic treatment with T for 4 months and two serial passages in mice. The prognostic value of AXL was evaluated in primary tumor cohort of HER2+ BC patients treated by standard guidelines (n=51). AXL was also explored in a cohort from PAMELA trial, a neoadjuvant phase II study. Patients were given lapatinib and T for 18 weeks (Luminal-HER2+ patients were additionally given hormonotherapy). Tumor samples were collected at baseline, day 14 and surgery (n= 96).
Results PDX resistant cell lines (TR1 and TR2) and resistant tumors (TR4) exhibit upregulation of AXL in comparison to the parental cells (PDX118). In vitro, the combination of an AXL inhibitor (TP-0903) + T resensitized resistant cells to T. In 3D organoids models from TR4, the combination decreased cell number in organoids more than both single agents. In TR4 in vivo model, TP-0903 + T completely abrogate tumor growth for more than 2 months.To validate our preclinical findings, we analyzed the level of AXL in our retrospectively cohort of patients. AXL level was significantly upregulated in patients who relapsed (p<0.0001). Patients with high levels of AXL had significantly shorter disease-free survival (DFS) and overall survival (OS) (log-rank p<0.0001; HR=15.78; 95% CI 3.89-19.80 for DFS; log-rank p=0.013; HR= 5.43; 95% CI 1.35-13.07 for OS). The median DFS and OS were 36 and 77 months in patients with high AXL level, whereas patients with low AXL level presented 92.5 and 92.5 months.To identify early molecular changes induced by dual HER2 blockade in patients with HER2+ disease, AXL was analyzed in PAMELA trial cohort. A significant increase of AXL was detected at day 14 compared to baseline (p=5.8e-20). This effect was observed in both luminal and non-luminal HER2+ BC patients and across all PAM50 subtypes. This rapid response suggests the implication of AXL as a mechanism of antiHER2 resistance. Levels of AXL decreased as long as treatment was on hold at surgery compared to day 14 (p=6.04e-14).
Conclusion AXL level was correlated with poor outcome of HER2+ BC patients. On our preclinical models, we showed that combination of TP-0903 and T abrogate tumor growth in acquired resistant models to T in PDXs. We postulate AXL as a promising new therapeutic strategy to overcome resistance to antiHER2 therapies
Citation Format: Anna Adam-Artigues, Raimundo Cervera, Enrique Javier Arenas, Fara Brasó-Maristany, Alex Martinez-Sabadell, Eduardo Tormo, Cristina Hernando, Maria Teresa Martinez, Soraya Simon, Jesús Poveda, Octavio Burgués, Ana Rovira, Federico Rojo, Joan Albanell, Begoña Bermejo, Ana Lluch, Pilar Eroles, Aleix Prat, Joaquin Arribas, Juan Miguel Cejalvo. AXL is a potential druggable target in trastuzumab resistance in HER2+ breast cancer patients abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1075.
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