Background: The addition of dendritic cell vaccines (DCV) to NAC could induce immune responses in those patients with residual disease (RD) by transforming the tumor microenvironment. Methods: Core diagnostic biopsies and surgical specimens from 80 patients (38 in the vaccinated group plus NAC (VG) and 42 in the control group (CG, treated only with NAC) were selected. We quantify TILs (CD8, CD4 and CD45RO) using immunohistochemistry and the automated cellular imaging system (ACIS III) in paired samples. Results: A CD8 rise in TNBC samples was observed after NAC plus DCV, changing from 4.48% in the biopsy to 6.70% in the surgical specimen, not reaching statistically significant differences (p = 0.11). This enrichment was seen in up to 67% of TNBC patients in the experimental arm as compared with the CG (20%). An association between CD8 TILs before NAC (4% cut-off point) and pathological complete response in the VG was found in the univariate and multivariate analysis (OR = 1.41, IC95% 1.05-1.90; p = 0.02, and OR = 2.0, IC95% 1.05-3.9; p = 0.03, respectively). Conclusion: Our findings suggest that patients with TNBC could benefit from the stimulation of the antitumor immune system by using DCV together with NAC.

Objectives: Immunostimulatory therapies using immune checkpoint blockers show clinical activity in a subset of glioblastoma (GBM) patients. Several inhibitory mechanisms play a relevant role in the immune response to GBM. With the objective of analyzing the tumor immune microenvironment and its clinical significance, we quantified several relevant immune biomarkers. Design: We studied 76 primary (non-recurrent) GBMs with sufficient clinical follow-up, including a subgroup of patients treated with a dendritic cell vaccine. The IDH-mutation, EGFR-amplification, and MGMT methylation statuses were determined. Several relevant immune biomarkers, including CD163, CD8, PD1, and PDL1, were quantified in representative selected areas by digital image analysis and semiquantitative evaluation. The percentage of each immune expression was calculated with respect to the total number of tumor cells. Results: All GBMs were wild-type IDH, with a subgroup of classical GBMs according to the EGFR amplification (44%). Morphologically, CD163 immunostained microglia and intratumor clusters of macrophages were observed. A significant direct correlation was found between the expression of CD8 and the mechanisms of lymphocyte immunosuppression, in such a way that higher values of CD8 were directly associated with higher values of CD163 (p < 0.001), PDL1 (0.026), and PD1 (0.007). In a multivariate analysis, high expressions of CD8+ (HR = 2.05, 95%CI (1.02-4.13), p = 0.034) and CD163+ cells (HR 2.50, 95%CI (1.29-4.85), p = 0.007), were associated with shorter survival durations. The expression of immune biomarkers was higher in the non-classical (non-EGFR amplified tumors) GBMs. Other relevant prognostic factors were age, receipt of the dendritic cell vaccine, and MGMT methylation status. Conclusions: In accordance with the inverse correlation between CD8 and survival and the direct correlation between effector cells and CD163 macrophages and immune-checkpoint expression, we postulate that CD8 infiltration could be placed in a state of anergy or lymphocytic inefficient activity. Furthermore, the significant inverse correlation between CD163 tissue concentration and survival explains the relevance of this type of immune cell when creating a strong immunosuppressive environment. This information may potentially be used to support the selection of patients for immunotherapy.

Malignant salivary gland tumors represent a challenge for pathologists due to their low frequency and morphologic overlap. In recent years machine learning techniques have been applied to the field of pathology to improve diagnostic performance. In the present work, we fitted a machine learning algorithm to approach the diagnosis of malignant salivary gland tumors. Twelve morphologic variables were scored across 115 samples representing the most commonly encountered malignant salivary gland tumors. The sample was randomly split into a discovery and validation set. A recursive partitioning algorithm was used to systematically screen and organize candidate variables into a classification tree using the discovery set. A cross-validation strategy was used to tune the algorithm hyperparameters. Inter-observer concordance was calculated by independent evaluation of 26 randomly selected cases. The five-tiered tree built, required the evaluation of 6 morphological variables. Basaloid appearance, presence of mucous cells, necrosis, cribriform pattern, clear cells and keratinization were selected by the algorithm to build the tree. This diagnostic tool correctly classified 89.9% and 84.6% of the samples in the discovery and validation sets respectively. Misclassification pattern was consistent between both sets. Misclassified tumors belonged to one of three histologic types: epithelial-myoepithelial, polymorphous and mucoepidermoid carcinomas. Other histotypes demonstrated perfect recall in both the discovery and validation sets. Overall inter-observer concordance was good, with median kappa scores between the expert evaluator and training pathologists being 0.81. Overall, our classification tool developed using a recursive partitioning algorithm can effectively guide the morphological approach to malignant salivary gland tumors.

Endometrial tumors show substantial heterogeneity in their immune microenvironment. This heterogeneity could be used to improve the accuracy of current outcome prediction tools. We assessed the immune microenvironment of 235 patients diagnosed with low-grade, early-stage endometrial cancer. Multiplex quantitative immunofluorescence was carried out to measure CD8, CD68, FOXP3, PD-1, and PD-L1 markers, as well as cytokeratin (CK), on tissue microarrays. Clustering results revealed five robust immune response patterns, each associated with specific immune populations, cell phenotypes, and cell spatial clustering. Most samples (69%) belonged to the immune-desert subtype, characterized by low immune cell densities. Tumor-infiltrating lymphocyte (TIL)-rich samples (4%) displayed high CD8(+) T-cell infiltration, as well as a high percentage of CD8/PD-1(+) cells. Immune-exclusion samples (19%) displayed the lowest CD8(+) infiltration combined with high PD-L1 expression levels in CK+ tumor cells. In addition, they demonstrated high tumor cell spatial clustering as well as increased spatial proximity of CD8(+)/PD-1(+) and CK/PD-L1(+) cells. FOXP3 and macrophage-rich phenotypes (3% and 4% of total samples) displayed relatively high levels of FOXP3(+) regulatory T-cells and CD68(+) macrophages, respectively. These phenotypes correlated with clinical outcomes, with immune-exclusion tumors showing an association with tumor relapse. When compared with prediction models built using routine pathological variables, models optimized with immune variables showed increased outcome prediction capacity (AUC = 0.89 versus 0.78) and stratification potential. The improved prediction capacity was independent of mismatch repair protein status and adjuvant radiotherapy treatment. Further, immunofluorescence results could be partially recapitulated using single-marker immunohistochemistry (IHC) performed on whole tissue sections. TIL-rich tumors demonstrated increased CD8(+) T-cells by IHC, while immune-exclusion tumors displayed a lack of CD8(+) T-cells and frequent expression of PD-L1 in tumor cells. Our results demonstrate the capability of the immune microenvironment to improve standard prediction tools in low-grade, early-stage endometrial carcinomas.

Simple Summary Low-grade, early-stage endometrial cancer (EC) is the most frequent malignant tumor of the uterine corpus. Our study aimed to assess dysregulated pathways in this specific subset of EC through proteomic analysis. We describe and validate the dysregulation of the SLIT/ROBO signaling pathway, as well as cellular death processes such as necroptosis and ferroptosis. We identify several immune-related pathways, with a dominance of innate immune response associated pathways. Our findings reveal the singular biology of low-grade, early-stage ECs and could guide future research in the field. Low-grade, early-stage endometrial carcinoma (EC) is the most frequent malignant tumor of the uterine corpus. However, the molecular alterations that underlie these tumors are far from being fully understood. The purpose of this study is to describe dysregulated molecular pathways from EC patients. Sixteen samples of tumor tissue and paired healthy controls were collected and both were subjected to mass spectrometry (MS)/MS proteomic analysis. Gene ontology and pathway analysis was performed to discover dysregulated pathways and/or proteins using different databases and bioinformatic tools. Dysregulated pathways were cross-validated in an independent external cohort. Cell signaling, immune response, and cell death-associated pathways were robustly identified. The SLIT/ROBO signaling pathway demonstrated dysregulation at the proteomic and transcriptomic level. Necroptosis and ferroptosis were cell death-associated processes aberrantly regulated, in addition to apoptosis. Immune response-associated pathways showed a dominance of innate immune responses. Tumor immune infiltrates measured by immunofluorescence demonstrated diverse lymphoid and myeloid populations. Our results suggest a role of SLIT/ROBO, necroptosis, and ferroptosis, as well as a prominent role of innate immune response in low-grade, early-stage EC. These results could guide future research in this group of tumors.

Glioblastoma is the most common primary central nervous system malignancy and has a poor prognosis. Standard first-line treatment, which includes surgery followed by adjuvant radio-chemotherapy, produces only modest benefits to survival1,2. Here, to explore the feasibility, safety and immunobiological effects of PD-1 blockade in patients undergoing surgery for glioblastoma, we conducted a single-arm phase II clinical trial (NCT02550249) in which we tested a presurgical dose of nivolumab followed by postsurgical nivolumab until disease progression or unacceptable toxicity in 30 patients (27 salvage surgeries for recurrent cases and 3¿cases of primary surgery for newly diagnosed patients). Availability of tumor tissue pre- and post-nivolumab dosing and from additional patients who did not receive nivolumab allowed the evaluation of changes in the tumor immune microenvironment using multiple molecular and cellular analyses. Neoadjuvant nivolumab resulted in enhanced expression of chemokine transcripts, higher immune cell infiltration and augmented TCR clonal diversity among tumor-infiltrating T lymphocytes, supporting a local immunomodulatory effect of treatment. Although no obvious clinical benefit was substantiated following salvage surgery, two of the three patients treated with nivolumab before and after primary surgery remain alive 33 and 28 months later.

The striatum (caudate nucleus, putamen and nucleus accumbens) is the main input structure of the basal ganglia. It receives cortical projections from the vast majority of the cortex, as well as from other subcortical structures such as the thalamus and amygdala. Its role in planning, preparation and execution of voluntary movements is known to be fine-tuned by the interaction between projection neurons and interneurons. Since the 1990s, it has been accepted that the proportion of interneurons increases phylogenetically, being about 5% in rodents and 26% in humans. However, these data have not been confirmed with unbiased techniques, such as stereology. In the present report, we have divided the human striatum into functional territories (associative, sensorimotor and limbic) and we have quantified the numerical density of all striatal neurons (using Nissl staining) in each area. Taking into account our past research on the estimation of striatal interneurons, we have calculated the proportion of interneurons in each territory. This value was on average 17.1% for the whole striatum, although interneurons were more abundant in the associative (21.9%) than in the sensorimotor (12.8%) and limbic (11.1%) aspects. Therefore, we demonstrate with unbiased stereology that the overall proportion of striatal interneurons is slightly lower than that reported in previous studies, and that it varies in the functional territories of this structure.