FGFR2-triggered autophagy and activation of Nrf-2 reduce breast cancer cell response to anti-ER drugs.

BACKGROUND: Genetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting strategies. Recent findings indicate that not only mutated FGFR is a driver of tumour progression but co-mutational landscapes and other markers should be also investigated. Autophagy has been recognized as one of the major mechanisms underlying the role of tumour microenvironment in promotion of cancer cell survival, and resistance to anti-ER drugs. The selective autophagy receptor p62/SQSTM1 promotes Nrf-2 activation by Keap1/Nrf-2 complex dissociation. Herein, we have analysed whether the negative effect of FGFR2 on BCa cell response to anti-ER treatment involves the autophagy process and/or p62/Keap1/Nrf-2 axis. METHODS: The activity of autophagy in ER-positive MCF7 and T47D BCa cell lines was determined by analysis of expression level of autophagy markers (p62 and LC3B) and monitoring of autophagosomes' maturation. Western blot, qPCR and proximity ligation assay were used to determine the Keap1/Nrf-2 interaction and Nrf-2 activation. Analysis of 3D cell growth in Matrigel® was used to assess BCa cell response to applied treatments. In silico gene expression analysis was performed to determine FGFR2/Nrf-2 prognostic value. RESULTS: We have found that FGFR2 signalling induced autophagy in AMPKα/ULK1-dependent manner. FGFR2 activity promoted dissociation of Keap1/Nrf-2 complex and activation of Nrf-2. Both, FGFR2-dependent autophagy and activation of Nrf-2 were found to counteract the effect of anti-ER drugs on BCa cell growth. Moreover, in silico analysis showed that high expression of NFE2L2 (gene encoding Nrf-2) combined with high FGFR2 expression was associated with poor relapse-free survival (RFS) of ER+ BCa patients. CONCLUSIONS: This study revealed the unknown role of FGFR2 signalling in activation of autophagy and regulation of the p62/Keap1/Nrf-2 interdependence, which has a negative impact on the response of ER+ BCa cells to anti-ER therapies. The data from in silico analyses suggest that expression of Nrf-2 could act as a marker indicating potential benefits of implementation of anti-FGFR therapy in patients with ER+ BCa, in particular, when used in combination with anti-ER drugs.

High tumour-infiltrating lymphocytes correlate with distinct gene expression profile and favourable survival in single hormone receptor-positive breast cancer.

Introduction: This study aimed to evaluate the impact of tumour-infiltrating lymphocytes (TILs) on the expression of immune-related genes and prognosis in single hormone receptor-positive breast cancer. Material and methods: Tumour-infiltrating lymphocytes were analysed according to the guidelines of the International TILs Working Group in a cohort of 206 patients with single hormone receptor-positive breast cancer. Of these, 44.7% were classified as ER+/PgR-/HER2-, 18.4% as ER+/PgR-/HER2+, 26.2% as ER-/PgR+/HER2-, and 10.7% as ER-/PgR+/HER2+. Moreover, in 52 samples the analysis of gene expression profiling was performed using nCounter technology. Results: Most cases (74.3%) showed at least 1% of stromal TILs, with a median of 4%, mean of 16.3%, and interquartile range of 0-20%. ER-/PgR+ tumours displayed significantly higher TILs density than ER+/PgR- cases (p < 0.001, Wilcoxon test), regardless of HER2 status. The abundance of TILs was positively associated with ER-/PgR+ phenotype, higher Ki-67, and higher grade, but not with age, tumour size, or regional and distant metastases at diagnosis. Additionally, in ER+/PgR- subgroup higher TILs were associated with HER2-positive status. Stromal TILs > 5% were associated with better survival in the whole group, but this effect was less prominent in ER-/PgR+ patients. We identified 50 differentially expressed genes (DEGs) between single hormone receptor-positive breast tumours with high and low TILs, including 39 up-regulated and 11 down-regulated genes in the high TILs group. Conclusions: The up-regulated expression of immune-related genes was consistent also among separately analysed single hormone receptor-positive groups (ER+/PgR- and ER-/PgR+).

Radiotherapy-induced dynamic changes in the lymphocyte-to-monocyte ratio in patients with laryngeal cancer indicate poor prognosis.

Aim: We hypothesized that markers of inflammation correlate with response to radiotherapy in patients with non-metastatic laryngeal cancer (LC). Our aim was to assess peripheral and local markers of inflammation including lymphocyte to monocyte ratio (LMR), neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), infiltrating CD8+ lymphocytes (TILsCD8), and programmed death 1 ligand (PD-L1) expression. Methods: We performed a retrospective single-center analysis of LC patients administered definitive (R-RT) or postoperative radiotherapy (PORT). The primary endpoint was overall survival (OS) in relation to peripheral and local inflammatory markers and their dynamic changes during RT. Results: Study group included 215 patients (R-RT, n=116; PORT, n=99). The baseline (t0) NLR and LMR were significantly correlated with OS in the R-RT group. In patients with high and low NLR at t0, the five-year OS was 33% and 56% (p=0.010) and in high and low LMR at t0, the five-year OS was 56% and 27% (p=0.003), respectively. The LMR increase during R-RT predicted better prognosis: the five-year OS in high and low LMR was 57% and 31% at t2 (after 2 weeks of RT) (p=0.015), 49% and 26% at t4 (p< 0.001), and 50% and 25% at t6 (p=0.013), respectively. Multivariable analysis shows that the worse performance status (p=0.003), the presence of nodal metastases (p=0.0001), and low baseline LMR (p=0.049) in the R-RT group, and the presence of nodal metastases (p=0.035) and completion treatment on time (p=0.042) in PORT group were associated with poor prognosis. The PD-L1 expression had no significant prognostic value in any of the examined patients. Conclusion: The baseline LMR and its dynamic changes during R-RT and baseline NLR are independent prognostic factors in patients with nonmetastatic LC. PD-L1 expression and number of TILsCD8 have no prognostic value in R-RT and PORT group.

FGFR2 Controls Growth, Adhesion and Migration of Nontumorigenic Human Mammary Epithelial Cells by Regulation of Integrin ß1 Degradation.

The role of fibroblast growth factor receptor 2 (FGFR2), an important mediator of stromal paracrine and autocrine signals, in mammary gland morphogenesis and breast cancer has been extensively studied over the last years. However, the function of FGFR2 signalling in the initiation of mammary epithelial oncogenic transformation remains elusive. Here, FGFR2-dependent behaviour of nontumorigenic model of mammary epithelial cells was studied. In vitro analyses demonstrated that FGFR2 regulates epithelial cell communication with extracellular matrix (ECM) proteins. Silencing of FGFR2 significantly changed the phenotype of cell colonies in three-dimensional cultures, decreased integrins α2, α5 and ß1 protein levels and affected integrin-driven processes, such as cell adhesion and migration. More detailed analysis revealed the FGFR2 knock-down-induced proteasomal degradation of integrin ß1. Analysis of RNA-seq databases showed significantly decreased FGFR2 and ITGB1 mRNA levels in breast tumour samples, when compared to non-transformed tissues. Additionally, high risk healthy individuals were found to have disrupted correlation profiles of genes associated with FGFR2 and integrin signalling, cell adhesion/migration and ECM remodelling. Taken together, our results strongly suggest that FGFR2 loss with concomitant integrin ß1 degradation is responsible for deregulation of epithelial cell-ECM interactions and this process may play an important role in the initiation of mammary gland epithelial tumorigenesis.

Glucose-6-phosphate dehydrogenase and 8-iso-prostaglandin F2α as potential predictors of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.

OBJECTIVE: Delayed cerebral ischemia (DCI) is a serious complication of aneurysmal subarachnoid hemorrhage (aSAH), which is responsible for significant death and disability. The dynamic balance between the production and elimination of reactive oxygen species (ROS) in patients with DCI is suspected be shifted to favor ROS formation. The authors assessed the relationship between F2-isoprostanes (F2-IsoPs), oxidative stress biomarkers, and glucose-6-phosphate dehydrogenase (G6PD), which are responsible for nicotinamide adenine dinucleotide phosphate (NADPH) production for glutathione system function, with post-aSAH DCI. METHODS: The authors assessed 45 aSAH patients for F2-IsoP and G6PD concentration using commercial ELISA on days 2, 4, and 6 after aSAH. The authors examined the correlation between plasma F2-IsoP and G6PD concentrations and clinical factors with DCI occurrence and aSAH outcome. RESULTS: Expectedly, the most important clinical predictors of DCI were Hunt and Hess grade and modified Fisher (mFisher) grade. Plasma F2-IsoP and G6PD concentrations were greater in aSAH patients than the control group (p < 0.01). F2-IsoP concentrations were greater and G6PD concentrations were lower in patients with DCI than those without (p < 0.01). Plasma F2-IsoP and G6PD concentrations on day 2 were correlated with DCI occurrence (p < 0.01). Plasma F2-IsoP concentrations on days 2 and 6 were correlated with outcome at 1 and 12 months (p < 0.01). CONCLUSIONS: Decreased G6PD indirectly informs the reduced antioxidant response, especially for the glutathione system. G6PD concentration was lower in patients with DCI than those without, which may explain the increased F2-IsoP concentrations. mFisher grade, plasma F2-IsoP concentration, and G6PD concentration on day 2 after aSAH, in combination, may serve as predictors of DCI. Further research is necessary to investigate the therapeutic utility of F2-IsoPs and antioxidants in clinical practice.

Immune checkpoint receptor VISTA on immune cells is associated with expression of T-cell exhaustion marker TOX and worse prognosis in renal cell carcinoma with venous tumor thrombus.

PURPOSE: The study aimed to determine the expression of VISTA and TOX within venous tumor thrombus and primary clear cell renal cell carcinoma (ccRCC) and to assess their prognostic value. METHODS: The study enrolled 82 patients with ccRCC and coexisting venous tumor thrombus treated radically from 2012 to 2019 in two tertiary centers. Tissue microarrays were prepared and stained with respective antibodies. The expression of markers was assessed separately on tumor cells (TCs) and/or tumor-associated immune cells (TAICs). RESULTS: TOX expression was positively correlated with the percentage of VISTA-positive TAICs in venous thrombus (p = 0.011), but not in the primary tumor (p = 0.674). High TOX expression was associated with a higher percentage of PD-L1-positive TAICs in both compartments (p = 0.001, p = 0.011, respectively). Positive expression of VISTA on TAICs was associated with PD-L1 expression on TCs (p = 0.005) and TAICs (p = 0.004) in the primary tumor, and only with PD-L1 on TAICs in thrombus (p = 0.006). The presence of VISTA-positive TAICs in venous thrombus was significantly more common in females (p = 0.034), and positively correlated with metastases (p = 0.028), and tumor necrosis (p = 0.013). The cases with VISTA-positive TAICs in venous tumor thrombi had significantly shorter OS than VISTA-negative cases (p = 0.041). CONCLUSION: For the first time, we demonstrated the expression of VISTA- and TOX-positive TAICs in the venous tumor thrombus. We found the association between immune checkpoint receptors and T cell exhaustion markers in both tumor mass and venous thrombus. Finally, we demonstrated that abundance of VISTA-positive TAICs in venous tumor thrombus correlates with worse outcomes in ccRCC.

Diagnostically misleading aberrant terminal deoxynucleotidyl transferase expression in germ cell tumors.

Terminal deoxynucleotidyl transferase (TdT) is a unique type of DNA polymerase predominantly expressed in precursor lymphoid cells and acute lymphoblastic leukemia. It participates in the junctional diversity of T-cell receptors and immunoglobulins. Recently, aberrant TdT expression was found in seminomas. Here, we evaluated the expression of TdT in our cohort of germ cell tumors (GCTs) with two anti-TdT antibody clones. We included 173 cases of testicular GCTs, 5 ovarian dysgerminomas, and one gonadoblastoma in the study. Tissue microarrays containing representative tumor samples were constructed and subsequently stained with anti-TdT monoclonal rabbit antibody EP266 (Dako) and TdT rabbit polyclonal antibody (Cell Marque). Expression was assessed with the H-score. No specific nuclear reaction was observed for the polyclonal anti-TdT antibody. The H-score values varied between the histological subtypes for the EP266 antibody. Positive nuclear staining was consistently seen in germ cell neoplasia in situ , seminoma, dysgerminoma, and embryonal carcinoma. Pure tumors had higher TdT H-scores than the mixed ones. Teratomas, yolk sac tumors, and choriocarcinomas were almost uniformly negative. Our study confirms that aberrant expression of TdT by testicular and ovarian GCTs exemplifies a potential diagnostic pitfall in histopathological diagnostics.

Alpha-smooth muscle actin-positive cancer-associated fibroblasts secreting osteopontin promote growth of luminal breast cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) have been shown to support tumor development in a variety of cancers. Different markers were applied to classify CAFs in order to elucidate their impact on tumor progression. However, the exact mechanism by which CAFs enhance cancer development and metastasis is yet unknown. METHODS: Alpha-smooth muscle actin (α-SMA) was examined immunohistochemically in intratumoral CAFs of nonmetastatic breast cancers and correlated with clinicopathological data. Four CAF cell lines were isolated from patients with luminal breast cancer (lumBC) and classified according to the presence of α-SMA protein. Conditioned medium (CM) from CAF cultures was used to assess the influence of CAFs on lumBC cell lines: MCF7 and T47D cells using Matrigel 3D culture assay. To identify potential factors accounting for promotion of tumor growth by α-SMAhigh CAFs, nCounter PanCancer Immune Profiling Panel (NanoString) was used. RESULTS: In luminal breast cancer, presence of intratumoral CAFs expressing high level of α-SMA (13% of lumBC group) correlated with poor prognosis (p = 0.019). In in vitro conditions, conditioned medium obtained from primary cultures of α-SMA-positive CAFs isolated from luminal tumors was observed to enhance growth of lumBC cell line colonies in 3D Matrigel, in contrast to CM derived from α-SMA-negative CAFs. Multigene expression analysis indicated that osteopontin (OPN) was overexpressed in α-SMA-positive CAFs in both clinical samples and in vitro models. OPN expression was associated with higher percentage of Ki67-positive cells in clinical material (p = 0.012), while OPN blocking in α-SMA-positive CAF-derived CM attenuated growth of lumBC cell line colonies in 3D Matrigel. CONCLUSIONS: Our findings demonstrate that α-SMA-positive CAFs might enhance tumor growth via secretion of OPN.

ERα36-High Cancer-Associated Fibroblasts as an Unfavorable Factor in Triple-Negative Breast Cancer.

Background: Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment (TME). Estrogen receptor alpha 36 (ERα36), the alternatively spliced variant of ERα, is described as an unfavorable factor when expressed in cancer cells. ERα can be expressed also in CAFs; however, the role of ERα36 in CAFs is unknown. Methods: Four CAF cultures were isolated from chemotherapy-naïve BC patients and characterized for ERα36 expression and the NanoString gene expression panel using isolated RNA. Conditioned media from CAF cultures were used to assess the influence of CAFs on triple-negative breast cancer (TNBC) cells using a matrigel 3D culture assay. Results: We found that ERα36high CAFs significantly induced the branching of TNBC cells in vitro (p < 0.001). They also produced a set of pro-tumorigenic cytokines compared to ERα36low CAFs, among which hepatocyte growth factor (HGF) was the main inducer of TNBC cell invasive phenotype in vitro (p < 0.001). Tumor stroma rich in ERα36high CAFs was correlated with high Ki67 expression (p = 0.041) and tumor-associated macrophages markers (CD68 and CD163, p = 0.041 for both). HGF was found to be an unfavorable prognostic factor in TCGA database analysis (p = 0.03 for DFS and p = 0.04 for OS). Conclusions: Breast cancer-associated fibroblasts represent distinct subtypes based on ERα36 expression. We propose that ERα36high CAFs could account for an unfavorable prognosis for TNBC patients.

Evaluation of PD-L1 (E1L3N, 22C3) expression in venous tumor thrombus is superior to its assessment in renal tumor in predicting overall survival in renal cell carcinoma.

BACKROUND: Renal cell carcinoma (RCC) frequently invades renal vein forming neoplastic thrombus. The expression of immune checkpoint receptors in RCC was addressed in multiple studies, but little is known about the expression and prognostic significance of programmed death ligand-1 in tumor thrombus. MATERIAL AND METHODS: The study aimed to evaluate the expression of PD-L1 within venous tumor thrombus and primary tumor using 2 independent antibody clones (22c3 and E1L3N) and to assess its value in predicting overall survival (OS) in the subgroup of patients with RCC and renal vein thrombus. RESULTS: Eighty-two patients with RCC and venous tumor thrombus that underwent nephrectomy were enrolled. The expression of PD-L1 was assessed utilizing tissue microarrays separately on tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs). The frequency of PD-L1 expression on TCs and TILs varied between tumor and thrombus compartments and was dependent on the antibody clone used. The expression of PD-L1 on TCs and/or TILs was associated with worse OS irrespectively of the antibody and the analyzed compartment. Nevertheless, the best prognostic performance was noted for the combined assessment of PD-L1 expression on TCs and TILs in venous tumor thrombus with the use of 22c3 antibody. The multivariable Cox regression model predicting OS incorporated PD-L1 22c3 in venous tumor thrombus (hazards ratio [HR] = 3.64, 95% confidence interval [CI] = 1.63-8.14, P = 0.002) and nodal status (HR = 2.88, 95% CI = 1.18-7.03, P = 0.02). CONCLUSIONS: PD-L1 may become a valuable tool for prognostic purposes in this specific subgroup of patients and be incorporated into the respective models qualifying for adjuvant treatment.

Higher platelet counts correlate to tumour progression and can be induced by intratumoural stroma in non-metastatic breast carcinomas.

BACKGROUND: Platelets support tumour progression. However, their prognostic significance and relation to circulating tumour cells (CTCs) in operable breast cancer (BrCa) are still scarcely known and, thus, merit further investigation. METHODS: Preoperative platelet counts (PCs) were compared with clinical data, CTCs, 65 serum cytokines and 770 immune-related transcripts obtained using the NanoString technology. RESULTS: High normal PC (hPC; defined by the 75th centile cut-off) correlated with an increased number of lymph node metastases and mesenchymal CTCs in the 70 operable BrCa patients. Patients with hPC and CTC presence revealed the shortest overall survival compared to those with no CTC/any PC or even CTC/normal PC. Adverse prognostic impact of hPC was observed only in the luminal subtype, when 247 BrCa patients were analysed. hPC correlated with high content of intratumoural stroma, specifically its phenotype related to CD8+ T and resting mast cells, and an increased concentration of cytokines related to platelet activation or even production in bone marrow (i.e. APRIL, ENA78/CXCL5, HGF, IL16, IL17a, MDC/CCL22, MCP3, MMP1 and SCF). CONCLUSIONS: Preoperative platelets evaluated alone and in combination with CTCs have prognostic potential in non-metastatic BrCa and define patients at the highest risk of disease progression, putatively benefiting from anti-platelet therapy.

Low Tumor-to-Stroma Ratio Reflects Protective Role of Stroma against Prostate Cancer Progression.

Tumor-to-stroma ratio (TSR) is a prognostic factor that expresses the relative amounts of tumor and intratumoral stroma. In this study, its clinical and molecular relevance was evaluated in prostate cancer (PCa). The feasibility of automated quantification was tested in digital scans of tissue microarrays containing 128 primary tumors from 72 PCa patients stained immunohistochemically for epithelial cell adhesion molecule (EpCAM), followed by validation in a cohort of 310 primary tumors from 209 PCa patients. In order to investigate the gene expression differences between tumors with low and high TSR, we applied multigene expression analysis (nCounter® PanCancer Progression Panel, NanoString) of 42 tissue samples. TSR scores were categorized into low (<1 TSR) and high (≥1 TSR). In the pilot cohort, 31 patients (43.1%) were categorized as low and 41 (56.9%) as high TSR score, whereas 48 (23.0%) patients from the validation cohort were classified as low TSR and 161 (77.0%) as high. In both cohorts, high TSR appeared to indicate the shorter time to biochemical recurrence in PCa patients (Log-rank test, p = 0.04 and p = 0.01 for the pilot and validation cohort, respectively). Additionally, in the multivariate analysis of the validation cohort, TSR predicted BR independent of other factors, i.e., pT, pN, and age (p = 0.04, HR 2.75, 95%CI 1.07-7.03). Our data revealed that tumors categorized into low and high TSR score show differential expression of various genes; the genes upregulated in tumors with low TSR score were mostly associated with extracellular matrix and cell adhesion regulation. Taken together, this study shows that high stroma content can play a protective role in PCa. Automatic EpCAM-based quantification of TSR might improve prognostication in personalized medicine for PCa.

Lost but Not Least-Novel Insights into Progesterone Receptor Loss in Estrogen Receptor-Positive Breast Cancer.

Estrogen receptor α (ERα) and progesterone receptor (PgR) are crucial prognostic and predictive biomarkers that are usually co-expressed in breast cancer (BC). However, 12-24% of BCs present ERα(+)/PgR(-) phenotype at immunohistochemical evaluation. In fact, BC may either show primary PgR(-) status (in chemonaïve tumor sample), lose PgR expression during neoadjuvant treatment, or acquire PgR(-) phenotype in local relapse or metastasis. The loss of PgR expression in ERα(+) breast cancer may signify resistance to endocrine therapy and poorer outcomes. On the other hand, ERα(+)/PgR(-) BCs may have a better response to neoadjuvant chemotherapy than double-positive tumors. Loss of PgR expression may be a result of pre-transcriptional alterations (copy number loss, mutation, epigenetic modifications), decreased transcription of the PGR gene (e.g., by microRNAs), and post-translational modifications (e.g., phosphorylation, sumoylation). Various processes involved in the down-regulation of PgR have distinct consequences on the biology of cancer cells. Occasionally, negative PgR status detected by immunohistochemical analysis is paradoxically associated with enhanced transcriptional activity of PgR that might be inhibited by antiprogestin treatment. Identification of the mechanism of PgR loss in each patient seems challenging, yet it may provide important information on the biology of the tumor and predict its responsiveness to the therapy.

Transcriptomic landscape of blood platelets in healthy donors.

Blood platelet RNA-sequencing is increasingly used among the scientific community. Aberrant platelet transcriptome is common in cancer or cardiovascular disease, but reference data on platelet RNA content in healthy individuals are scarce and merit complex investigation. We sought to explore the dynamics of platelet transcriptome. Datasets from 204 healthy donors were used for the analysis of splice variants, particularly with regard to age, sex, blood storage time, unit of collection or library size. Genes B2M, PPBP, TMSB4X, ACTB, FTL, CLU, PF4, F13A1, GNAS, SPARC, PTMA, TAGLN2, OAZ1 and OST4 demonstrated the highest expression in the analysed cohort, remaining substantial transcription consistency. CSF3R gene was found upregulated in males (fold change 2.10, FDR q < 0.05). Cohort dichotomisation according to the median age, showed upregulated KSR1 in the older donors (fold change 2.11, FDR q < 0.05). Unsupervised hierarchical clustering revealed two clusters which were irrespective of age, sex, storage time, collecting unit or library size. However, when donors are analysed globally (as vectors), sex, storage time, library size, the unit of blood collection as well as age impose a certain degree of between- and/or within-group variability. Healthy donor platelet transcriptome retains general consistency, with very few splice variants deviating from the landscape. Although multidimensional analysis reveals statistically significant variability between and within the analysed groups, biologically, these changes are minor and irrelevant while considering disease classification. Our work provides a reference for studies working both on healthy platelets and pathological conditions affecting platelet transcriptome.

Activation of epithelial-mesenchymal transition process during breast cancer progression - the impact of molecular subtype and stromal composition.

Breast cancer (BC) is a heterogeneous disease with different molecular subtypes, which can be defined by oestrogen (ER), progesterone (PR) and human epidermal growth factor (HER2) receptors' status as luminal, HER2+ and triple negative (TNBC). Molecular subtypes also differ in their epithelial-mesenchymal phenotype, which might be related to their aggressiveness, as activation of the epithelial-mesenchymal transition (EMT) is linked with increased ability of cancer cells to survive and metastasize. Nevertheless, the reverse process of mesenchymal-epithelial transition was shown to be required to sustain metastatic colonization. In this study we aimed to analyse activation of the EMT process in primary tumours (PT), which have (N+) or have not (N-) colonized the lymph nodes, as well as the lymph nodes metastases (LNM) themselves in 88 BC patients. We showed that luminal N- PT have the lowest activation of the EMT process (27%), in comparison to N+ PT (48%, p=0.06). On the other hand, TNBC do not show statistically significant EMT activation at the stage before lymph colonization (N-, 83%) and after colonization of the lymph nodes (N+, 63%, p=0.58). TNBC are also the least plastic (unable to change the EMT phenotype) in terms of turning EMT on or off between matched PT and LNM (0% EMT plasticity in TNBC vs 36% plasticity in luminal tumours). Moreover, in TNBC activation of EMT was correlated with increased cell division rate of the PT- in mesenchymal TNBC PT median Ki-67 was 45% in comparison to 10% in epithelial TNBC PT (p=0.002), whereas in PT of luminal subtypes Ki-67 did not differ between epithelial and mesenchymal phenotypes. Profiling of immunotranscriptome of epithelial and mesenchymal luminal BC with Nanostring technology revealed that N- PT with epithelial phenotype were enriched in inflammatory response signatures, whereas N+ mesenchymal cancers showed elevated MHC class II antigen presentation. Overall, activation of EMT changes during cancer progression and metastatic colonization of the lymph nodes depending on the PT molecular subtype and is related to differences in stromal signatures. Activation of EMT is associated with colonizing phenotype in luminal PT and proliferative phenotype of TNBC.

Combined Assessment of Immune Checkpoint Regulator VISTA on Tumor-Associated Immune Cells and Platelet-to-Lymphocyte Ratio Identifies Advanced Germ Cell Tumors with Higher Risk of Unfavorable Outcomes.

In the current study, we aimed to investigate whether expression of immune checkpoint proteins (V-domain Ig suppressor of T cell activation (VISTA) and programmed death-ligand 1 (PD-L1)) and markers of systemic inflammation could predict progression/relapse and death in the cohort of 180 patients with testicular germ-cell tumors (GCTs). Expression of PD-L1 and VISTA was assessed by immunohistochemistry utilizing tissue microarrays. To estimate systemic inflammation neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR) were calculated. We found high PD-L1 and VISTA expression on tumor-associated immune cells (TAICs) in 89 (49.44%) and 63 (37.22%) of GCTs, respectively, whereas tumor cells besides trophoblastic elements were almost uniformly negative. High PD-L1 was associated with seminomatous histology and lower stage. Relapses in stage I patients occurred predominantly in cases with low numbers of PD-L1 and VISTA-expressing TAICs. In stage II/III disease, the combination of low VISTA-expressing TAICs and high PLR was identified as predictor of shorter event-free survival (HR 4.10; 1.48-11.36, p = 0.006) and overall survival (HR 15.56, 95% CI 1.78-135.51, p = 0.001) independently of tumor histology and location of metastases. We demonstrated that the assessment of immune checkpoint proteins on TAICs may serve as a valuable prognostic factor in patients with high-risk testicular GCTs. Further study is warranted to explore the predictive utility of these biomarkers in GCTs.

Reduced expression of innate immunity-related genes in lymph node metastases of luminal breast cancer patients.

Immune system plays a dual role in cancer by either targeting or supporting neoplastic cells at various stages of disease, including metastasis. Yet, the exact immune-related transcriptome profiles of primary tumours (PT) and lymph node metastases (LNM) and their evolution during luminal breast cancer (BCa) dissemination remain undiscovered. In order to identify the immune-related transcriptome changes that accompany lymphatic spread, we analysed PT-LNM pairs of luminal BCa using NanoString technology. Decrease in complement C3-one of the top-downregulated genes, in LNM was validated at the protein level using immunohistochemistry. Thirty-three of 360 analysed genes were downregulated (9%), whereas only 3 (0.8%) upregulated in LNM when compared to the corresponding PT. In LNM, reduced expression was observed in genes related to innate immunity, particularly to the complement system (C1QB, C1S, C1R, C4B, CFB, C3, SERPING1 and C3AR1). In validation cohort, complement C3 protein was less frequently expressed in LNM than in PT and it was associated with worse prognosis. To conclude, local expression of the complement system components declines during lymphatic spread of non-metastatic luminal BCa, whilst further reduction of tumoral complement C3 in LNM is indicative for poor survival. This points to context-dependent role of complement C3 in BCa dissemination.

EGFR as a stable marker of prostate cancer dissemination to bones.

BACKGROUND: Prostate cancer (PCa) is among the most commonly diagnosed malignancies in men. Although 5-year survival in patients with localised disease reaches nearly 100%, metastatic disease still remains incurable. Therefore, there is a need for markers indicating metastatic dissemination. METHODS: EGFR overexpression (EGFRover) was tracked in 1039 primary tumours, circulating tumour cells from 39 d'Amico high-risk patients and metastatic samples from 21 castration-resistant PCa cases. EGFR status was compared to clinical parameters and multiple molecular factors were assessed using immunohistochemistry and gene ontology analysis. The functional aspect of EGFR was evaluated by plating PC-3 cells on soft and rigid matrices. RESULTS: EGFRover was found in 14% of primary tumours, where it was associated with shorter metastasis-free survival and was an independent indicator of worse overall survival. EGFRover correlated with a pro-migratory and pro-metastatic phenotype of tumour cells as well as rich collagen fibre content. All circulating tumour cells (detected in 13% of cases) were positive for EGFR, independent of their EMT-related phenotype. EGFRover was more prevalent in castration-resistant bone metastases (29% of patients) and supported growth of human PCa cells on rigid matrices mimicking bone stiffness. CONCLUSIONS: EGFRover is a stable, EMT-independent marker of PCa disseminating to rigid organs, preferentially bones.

microRNA Expression Profile in Single Hormone Receptor-Positive Breast Cancers is Mainly Dependent on HER2 Status-A Pilot Study.

Estrogen (ER) and progesterone (PgR) receptors and HER2 are crucial in the assessment of breast cancer specimens due to their prognostic and predictive significance. Single hormone receptor-positive breast cancers are less common and their clinical course is less favorable than ER(+)/PgR(+) tumors. Their molecular features, especially microRNA (miRNA) profiles, have not been investigated to date. Tumor specimens from 36 chemonaive breast cancer patients with known ER and PgR status (18 ER(+)/PgR(-) and 18 ER(-)/PgR(+) cases) were enrolled to the study. The expression of 829 miRNAs was evaluated with nCounter Human v3 miRNA expression Assay (NanoString). miRNAs differentiating between ER/PgR/HER2 phenotypes were selected based on fold change (FC) calculated for the mean normalized counts of each probe in compared groups. The differences were estimated with Student's T-test or Two-Way ANOVA (considering also the HER2 status). The results were validated using The Cancer Genome Atlas (TCGA) dataset. Following quality control of raw data, fourcases were excluded due to low sample quality, leaving 14 ER(+)/PgR(-) and 18 ER(-)/PgR(+) cases. After correction for multiple comparisons, we did not find miRNA signature differentiating between ER(-)/PgR(+) and ER(+)/PgR(-) breast cancers. However, a trend for differing expression (p-value ≤ 0.05; FDR > 0.2; ANOVA) in eight miRNAs was observed. The ER(+)/PgR(-) group demonstrated elevated levels of four miRNAs-miR-30a-5p, miR-29c-3p, miR-141-3p and miR-423-5p-while the ER(-)/PgR(+) tumors were enriched in another four miRNAs-miR-514b-5p, miR-424-5p, miR-495-3p, and miR-92a-3p. For one of the miRNAs-miR-29c-3p-the association with the ER(+)/PgR(-) phenotype was confirmed in the TCGA cohort (p-value = 0.024; T-test). HER2 amplification/overexpression in the NanoString cohort was related to significant differences observed in 33 miRNA expression levels (FDR ≤ 0.2; ANOVA). The association with HER2 status was confirmed in the TCGA cohort for four miRNAs (miR-1180-3p, miR-223-3p, miR-30d-5p, and miR-195-5p). The main differences in miRNA expression amongst single hormone receptor-positive tumors were identified according to their HER2 status. However, ER(+)/PgR(-) cases tended to express higher levels of miRNAs associated with ER-positivity (miR-30a-5p, miR-29c-3p, miR-141-3p), whereas ER(-)/PgR(+) cancers showed elevated levels of miRNAs characteristic for double- and triple-negative tumors (miR-92a-3p, miR-424-5p). Further studies are necessary to comprehensively analyze miRNA signatures characteristic of ER(-)/PgR(+) and ER(+)/PgR(-) tumors.

Liquid biopsy for minimally invasive heart transplant monitoring: a pilot study.

BACKGROUND: Heart transplantation allows for a long-term management of patients with end-stage heart failure. After the surgery, organ rejection is monitored with endomyocardial biopsy, which is an invasive, but not always informative procedure. Therefore, there is a pressing need for a new, safe, yet reliable, diagnostic method. Here, we present a pilot study confronting liquid biopsy based on donor-specific cell-free DNA with the protocol endomyocardial biopsy. METHODS: The study was performed on 21 blood samples matched with endomyocardial biopsy (graded according to acute cellular rejection scale) from nine patients after heart transplantation. Genotyping was performed on genomic DNA from donors and recipients for 10 single-nucleotide polymorphisms (SNPs). Cell-free DNA isolated from plasma was analysed with digital droplet PCR to detect donor-specific alleles. RESULTS: From 21 analysed endomyocardial biopsies, 4 were graded as 0R and 17 as 1R. Liquid biopsy was successfully performed in each sample for all informative SNPs (median of 3 per patient). We observed a high homogeneity of the results between SNPs in each sample (interclass correlation coefficient of >0.9). CONCLUSIONS: There is a undeniable need for an alternative, non-invasive diagnostic procedure of early transplant rejection and investigation of donor-derived cell-free DNA seems to be the promising choice. The very high sensitivity is particularly enticing to consider liquid biopsy as a potential screening tool. Its minimal invasiveness may allow for more frequent examination and, thus, tighter monitoring. The reliable assessment of its clinical utility requires an adequately powered and properly designed multicentre study.