Oxidative stress-induced FAK activation contributes to uterine serous carcinoma aggressiveness.

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer (EC), characterized by its high propensity for metastases. In fact, while endometrioid endometrial carcinoma (EEC), which accounts for 85% of EC, presents a good prognosis, USC is the most frequently fatal. Herein, we used for the first time a peptide-based tyrosine-kinase-activity profiling approach to quantify the changes in tyrosine kinase activation between USC and EEC. Among the tyrosine kinases highly activated in USC, we identified focal adhesion kinase (FAK). We conducted mechanistic studies using cellular models. In a USC cell line, targeting FAK either by inhibitors PF-573228 and defactinib (VS-6063) or by gene silencing limits 3D cell growth and reduces cell migration. Moreover, results from our studies suggest that oxidative stress is increased in USC tumors compared to EEC ones. Reactive oxygen species (ROS) induce tyrosine phosphorylation of FAK and a concomitant tyrosine phosphorylation of paxillin, a mediator of FAK signal transduction. Mechanistically, by tracking hundreds of individual cells per condition, we show that ROS increased cell distance and migration velocity, highlighting the role of ROS-FAK-PAX signaling in cell migration. Both defactinib and ROS scavenger N-acetylcysteine (NAC) revert this effect, pointing toward ROS as potential culprits for the increase in USC cell motility. A proof of concept of the role of FAK in controlling cell growth was obtained in in vivo experiments using cancer-tissue-originated spheroids (CTOS) and a patient-derived orthotopic xenograft model (orthoxenograft/PDOX). Defactinib reduces cell proliferation and protein oxidation, supporting a pro-tumoral antioxidant role of FAK, whereas antioxidant NAC reverts FAK inhibitor effects. Overall, our data points to ROS-mediated FAK activation in USC as being responsible for the poor prognosis of this tumor type and emphasize the potential of FAK inhibition for USC treatment.

Generation and Integrated Analysis of Advanced Patient-Derived Orthoxenograft Models (PDOX) for the Rational Assessment of Targeted Therapies in Endometrial Cancer.

Clinical management of endometrial cancer (EC) is handicapped by the limited availability of second line treatments and bona fide molecular biomarkers to predict recurrence. These limitations have hampered the treatment of these patients, whose survival rates have not improved over the last four decades. The advent of coordinated studies such as The Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA_UCEC) has partially solved this issue, but the lack of proper experimental systems still represents a bottleneck that precludes translational studies from successful clinical testing in EC patients. Within this context, the first study reporting the generation of a collection of endometrioid-EC-patient-derived orthoxenograft (PDOX) mouse models is presented that is believed to overcome these experimental constraints and pave the way toward state-of-the-art precision medicine in EC. The collection of primary tumors and derived PDOXs is characterized through an integrative approach based on transcriptomics, mutational profiles, and morphological analysis; and it is demonstrated that EC tumors engrafted in the mouse uterus retain the main molecular and morphological features from analogous tumor donors. Finally, the molecular properties of these tumors are harnessed to assess the therapeutic potential of trastuzumab, a human epidermal growth factor receptor 2 (HER2) inhibitor with growing interest in EC, using patient-derived organotypic multicellular tumor spheroids and in vivo experiments.

Metabolomic Analysis Points to Bioactive Lipid Species and Acireductone Dioxygenase 1 (ADI1) as Potential Therapeutic Targets in Poor Prognosis Endometrial Cancer.

Metabolomic profiling analysis has the potential to highlight new molecules and cellular pathways that may serve as potential therapeutic targets for disease treatment. In this study, we used an LC-MS/MS platform to define, for the first time, the specific metabolomic signature of uterine serous carcinoma (SC), a relatively rare and aggressive variant of endometrial cancer (EC) responsible for 40% of all endometrial cancer-related deaths. A metabolomic analysis of 31 ECs (20 endometrial endometrioid carcinomas (EECs) and 11 SCs) was performed. Following multivariate statistical analysis, we identified 232 statistically different metabolites among the SC and EEC patient samples. Notably, most of the metabolites identified (89.2%) were lipid species and showed lower levels in SCs when compared to EECs. In addition to lipids, we also documented metabolites belonging to amino acids and purine nucleotides (such as 2-Oxo-4-methylthiobutanoic acid, synthesised by acireductone dioxygenase 1 (ADI1) enzyme), which showed higher levels in SCs. To further investigate the role of ADI1 in SC, we analysed the expression protein levels of ADI1 in 96 ECs (67 EECs and 29 SCs), proving that the levels of ADI1 were higher in SCs compared to EECs. We also found that ADI1 mRNA levels were higher in p53 abnormal ECs compared to p53 wild type tumours. Furthermore, elevated ADI1 mRNA levels showed a statistically significant negative correlation with overall survival and progression-free survival among EEC patients. Finally, we tested the ability of ADI1 to induce migration and invasion capabilities in EC cell lines. Altogether, these results suggest that ADI1 could be a potential therapeutic target in poor-prognosis SCs and other Ecs with abnormal p53 expression.

Usefulness of urinary biomarkers to estimate the interstitial fibrosis surface in diabetic nephropathy with normal kidney function.

BACKGROUND: Kidney biopsies of patients with diabetic nephropathy (DN) and normal kidney function may exhibit interstitial fibrosis (IF) without reduction of glomerular filtration rate (GFR) because of hyperfiltration. The aim of our study was to analyse the performance of a set of biomarkers of tubular injury to estimate the extent of IF in patients with DN and normal kidney function. METHODS: This cross-sectional study included 118 adults with DN diagnosed by kidney biopsy and GFR ≥90 mL/min/1.73 m2 and a control group of healthy subjects. We measured the urinary excretion of monocyte chemoattractant protein-1 (MCP-1) neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), liver-type fatty acid-binding protein (L-FABP), ß2-microglobulin and dickkopf-3 protein (DKK-3) at the time of kidney biopsy. GFR was measured by chromium-51 labeled ethylenediamine tetraacetic acid (Cr-EDTA) (measured GFR). IF was quantified using a quantitative morphometric procedure. Predictive multivariate models were developed to estimate the IF surface. RESULTS: Patients with DN showed significantly higher levels of DKK-3, MCP-1 and L-FABP and significantly lower levels of epidermal growth factor (EGF) than healthy controls. There were no significant between-group differences in the levels of ß2-microglobulin, KIM-1 or NGAL. IF was negatively associated with EGF and positively with age, proteinuria, MCP-1, DKK-3 and L-FABP, but not with ß2-microglobulin, KIM-1, NGAL or GFR. The best model to predict IF surface accounted for 59% of its variability and included age, proteinuria, EGF, DKK-3 and MCP-1. CONCLUSIONS: Our study provides a model to estimate the IF in DN that can be useful to assess the progression of IF in patients with normal kidney function.

CD44-negative parietal-epithelial cell staining in minimal change disease: association with clinical features, response to corticosteroids and kidney outcome.

BACKGROUND: Activation of parietal-epithelial cells (PECs) with neo-expression of CD44 has been found to play a relevant role in the development of focal and segmental glomerulosclerosis (FSGS). The aim of this study was to analyse whether the expression of CD44 by PECs in biopsies of minimal change disease (MCD) is associated with the response to corticosteroids, with kidney outcomes and/or can be considered an early sign of FSGS. METHODS: This multicentric, retrospective study included paediatric and adult patients with MCD. Demographic, clinical and biochemical data were recorded, and biopsies were stained with anti-CD44 antibodies. The association between PECs, CD44 expression and the response to corticosteroids, and kidney outcomes were analysed using logistic, Kaplan-Meier and Cox regression analyses. RESULTS: A total of 54 patients were included: 35 (65%) <18 years and 19 (35%) adults. Mean follow-up was 68.3 ± 37.9 months. A total of 19/54 patients (35.2%) showed CD44-positive staining. CD44-positive patients were younger (14.5 ± 5 versus 21.5 ± 13, P = 0.006), and showed a higher incidence of steroid-resistance [11/19 (57.8%) versus 7/35 (20%), P = 0.021; odds ratio: 5.5 (95% confidence interval 1.6-18), P = 0.007] and chronic kidney disease [9/19 (47.3%) versus 6/35 (17.1%), P = 0.021; relative risk: 3.01 (95% confidence interval 1.07-8.5), P = 0.037]. Follow-up re-biopsies of native kidneys (n = 18), identified FSGS lesions in 10/12 (83.3%) of first-biopsy CD44-positive patients versus 1/6 (16.7%) of first-biopsy CD44-negative patients (P = 0.026). CONCLUSIONS: In patients with a light microscopy pattern of MCD, CD44-positive staining of PECs is associated with a higher prevalence of steroid resistance and worse kidney outcomes, and can be considered an early sign of FSGS.

ARID1A-deficient cells require HDAC6 for progression of endometrial carcinoma.

AT-rich interactive domain-containing protein 1A (ARID1A) loss-of-function mutation accompanied by a loss of ARID1A protein expression is frequently observed in endometrial carcinomas. However, the molecular mechanisms linking these genetic changes to the altered pathways regulating tumour initiation, maintenance and/or progression remain poorly understood. Thus, the main aim of this study was to analyse the role of ARID1A loss of function in endometrial tumorigenesis. Here, using different endometrial in vitro and in vivo models, such as tumoral cell lines, 3D primary cultures and metastatic or genetically modified mouse models, we show that altered expression of ARID1A is not enough to initiate endometrial tumorigenesis. However, in an established endometrial cancer context, ARID1A loss of function accelerates tumoral progression and metastasis through the disruption of the G2/M cell cycle checkpoint and ATM/ATR-mediated DNA damage checkpoints, increases epithelial cell proliferation rates and induces epithelial mesenchymal transition through the activation of histone deacetylase 6 (HDAC6). Next, we demonstrated that the inhibition of HDAC6 function, using the HDAC6-specific inhibitor ACY1215 or by transfection with HDAC6 short hairpin RNA (shRNA), can reverse the migratory and invasive phenotype of ARID1A-knockdown cells. Further, we also show that inhibition of HDAC6 activity causes an apoptotic vulnerability to etoposide treatments in ARID1A-deficient cells. In summary, the findings exposed in this work indicate that the inhibition of HDAC6 activity is a potential therapeutic strategy for patients suffering from ARID1A-mutant endometrial cancer diagnosed in advanced stages.

Differential Immunoexpression of BRAF/V600E, Senescence Markers, PTEN, and T-type Calcium Channels in Acquired Naevi According to their Histopathological and Dermoscopic Classification.

BRAF/V600E mutation and other cell growth/growth-control mechanisms are involved in naevogenesis and melanomagenesis. Immunoexpression of BRAF/V600E and other molecules (p16, phosphatase and tensin homologue (PTEN), Ki67, hTERT and Cav3.1 and 3.2 calcium channels) were investigated in 80 histopatho-logically and dermoscopically classified acquired naevi. Regarding BRAF/V600E, dysplastic naevi showed lower immunostaining than common naevi, which was significant in comparison with intradermal naevi, which showed the highest BRAF/V600E histoscore. Junctional naevi showed the lowest BRAF/V600E levels. Globular/cobblestone and reticular dermoscopic patterns were consistently associated with high and low BRAF/V600E immunoexpression, respectively, but Zalaudek's peripheral globule pattern (CR/PG) showed the highest BRAF/V600E immunoexpression. Among global patterns, the previously not investigated multicomponent pattern showed the lowest BRAF/V600E immunoexpression. Regarding the remaining biomarkers, new immunohistochemical features were found, in particular p16 and PTEN low expression in multicomponent pattern; and Ki67, hTERT and Cav.3.1 high expression in CR/PG. In conclusion, histopathology and dermoscopy provide complementary information regarding the biology of melanocytic naevi.

BRAFV600E Mutant Allele Frequency (MAF) Influences Melanoma Clinicopathologic Characteristics.

BACKGROUND: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. METHODS: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. RESULTS: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. CONCLUSIONS: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.

M-TRAP: Safety and performance of metastatic tumor cell trap device in advanced ovarian cancer patients.

OBJECTIVE: Despite radical surgery and chemotherapy, most patients with ovarian cancer die due to disease progression. M-Trap is an implantable medical device designed to capture peritoneal disseminated tumor cells with the aim to focalize the disease. This trial analyzed the safety and performance of the device. METHODS: This first-in-human prospective, multi-center, non-blinded, single-arm study enrolled 23 women with high-grade serous advanced ovarian cancer. After primary or interval debulking surgery, 3 M-Trap devices were placed in the peritoneum of the abdominal cavity. 18-months post-implantation or at disease progression, devices were initially removed by laparoscopy. The primary safety endpoint was freedom from device and procedure-related major adverse events (MAEs) through 6-months post-implantation compared to an historical control. The primary performance endpoint was histopathologic evidence of tumor cells capture. RESULTS: Only one major adverse event was attributable to the device. 18 women were free of device and procedure related MAEs (78.3%). However, the primary safety endpoint was not achieved (p = 0.131), primarily attributable to the greater surgical complexity of the M-Trap patient population. 62% of recurrent patients demonstrated tumor cell capture in at least one device with a minimal tumor cell infiltration. No other long-term device-related adverse events were reported. The secondary performance endpoint demonstrated a lack of disease focalization. CONCLUSIONS: The M-Trap technology failed to meet its primary safety objective, although when adjusted for surgical complexity, the study approved it. Likewise, the devices did not demonstrate the anticipated benefits in terms of tumor cell capture and disease focalization in recurrent ovarian cancer.

The cutoff for estrogen and progesterone receptor expression in endometrial cancer revisited: a European Network for Individualized Treatment of Endometrial Cancer collaboration study.

There is no consensus on the cutoff for positivity of estrogen receptor (ER) and progesterone receptor (PR) in endometrial cancer (EC). Therefore, we determined the cutoff value for ER and PR expression with the strongest prognostic impact on the outcome. Immunohistochemical expression of ER and PR was scored as a percentage of positive EC cell nuclei. Cutoff values were related to disease-specific survival (DSS) and disease-free survival (DFS) using sensitivity, specificity, and multivariable regression analysis. The results were validated in an independent cohort. The study cohort (n = 527) included 82% of grade 1-2 and 18% of grade 3 EC. Specificity for DSS and DFS was highest for the cutoff values of 1-30%. Sensitivity was highest for the cutoff values of 80-90%. ER and PR expression were independent markers for DSS at cutoff values of 10% and 80%. Consequently, three subgroups with distinct clinical outcomes were identified: 0-10% of ER/PR expression with, unfavorable outcome (5-year DSS = 75.9-83.3%); 20-80% of ER/PR expression with, intermediate outcome (5-year DSS = 93.0-93.9%); and 90-100% of ER/PR expression with, favorable outcome (5-year DSS = 97.8-100%). The association between ER/PR subgroups and outcomes was confirmed in the validation cohort (n = 265). We propose classification of ER and PR expression based on a high-risk (0-10%), intermediate-risk (20-80%), and low-risk (90-100%) group.

Developing indicators for quality assurance in cytopathology. Catalan Society of Cytopathology.

BACKGROUND: Quality control in cytology must be established through reliable and easily measurable indicators. METHODS: From the Catalan Society of Cytopathology a group of experts has been established to write a document with 13 indicators that cover the entire cytological process, based on its Cytopathology Quality Guide. It has been elaborated through guides and documents with scientific evidence and DELPHI methodology in order to reach a structured consensus on the opinions of a group of experts. RESULTS: Thirteen indicators, covering all the cytologic process are expressed in worksheets specifying all their characteristics. CONCLUSION: This document allows the control of all stages of the cytological process.

An olaparib window-of-opportunity trial in patients with early-stage endometrial carcinoma: POLEN study.

OBJECTIVE: Olaparib is a potent inhibitor of poly(ADP-ribose) polymerase (PARP)-1, 2, and 3 with potential activity in endometrial cancer (EC). METHODS: In this window-of-opportunity trial, women with operable type 1 EC received olaparib oral tablets (300mg) twice daily for 28days before surgery. The primary objective was to evaluate the effects of olaparib on EC in tissue samples taken at baseline and at treatment completion. Signal of activity was defined as significant changes in the expression of the cell cycle-related proteins cyclin D1, Ki67, and cleaved caspase-3. RESULTS: A total of 31 patients were included in the biomarker analysis. The median time of olaparib exposure was 24 days (1-39). Significant inhibition was found for cyclin D1 (p < 0.01), but not for Ki67 and active caspase 3 immunostaining. PARP-1 levels positively correlated with cyclin D1 levels (rho = 0.661, p = 0.0001). Both PARP-1 and cyclin D1 levels were significantly lower (p = 0.022 and p = 0.004, respectively) in patients with ARID1A[-] tumors than ARID1A[+] tumors. A significant relationship between plasma olaparib concentrations and decreased GLUT1 activity was observed (r = -0.5885; p < 0.05). Drug-related toxicity consisted mostly of gastrointestinal and grade 1 or 2 adverse events. CONCLUSIONS: Olaparib reduced expression of cyclin D1, which positively correlated with PARP-1 levels. This effect was more evident in ARID1A-deficient tumors. Olaparib further induced inhibition of GLUT1 plasma activity. Our findings could have noteworthy implications in predicting which patients with EC would benefit from olaparib-based strategies.

Biomimetic device and foreign body reaction cooperate for efficient tumour cell capture in murine advanced ovarian cancer.

Metastasis is facilitated by the formation of pre-metastatic niches through the remodelling of the extracellular matrix (ECM) promoted by haematopoietic and stromal cells. The impact of these primed sites is pronounced for intraperitoneal metastases, where the cavity-exposed ECM supports the attachment of the disseminating tumour cells. Likewise, implantation of biomaterial scaffolds influences metastatic progression systemically through a foreign body reaction (FBR). In this study, we integrated the concept of creating an artificial niche to capture tumour cells actively disseminating in the peritoneal cavity with a therapeutic strategy modulating the interactions of metastatic cells with the ECM. The aim was to transform a disseminated disease into a focal disease. For this, we designed and developed a 'biomimetic' ECM composed of a nonresorbable three-dimensional scaffold with collagen coating and characterized the FBR to the implanted biomaterial. We also analysed the safety of the implanted devices and their ability to capture tumour cells in different murine preclinical models of advanced ovarian cancer. Implantation of the biomimetic devices resulted in an initial inflammatory reaction that transformed progressively into a fibrous connective tissue response. The adhesive capabilities of the scaffold were improved with the ancillary effect of the FBR and showed clinical utility in terms of the efficacy of capture of tumour cells, disease focalization and survival benefit. These results demonstrated the performance and safety of this 'biomimetic' ECM in preclinical models of advanced ovarian cancer. Translated into the clinical setting, this new therapeutic strategy represents the possibility for control of peritoneal carcinomatosis upon primary ovarian debulking surgery and to expand the percentage of patients who are candidates for second rescue surgeries at the time of relapse.

Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals.

The causal mechanism for cancer predisposition in Lynch-like syndrome (LLS) remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive (epi)genetic analysis. One hundred and fifteen LLS patients harboring MMR-deficient tumors and no germline MMR mutations were included. Mutational analysis of 26 colorectal cancer (CRC)-associated genes was performed. Pathogenicity of MMR variants was assessed by splicing and multifactorial likelihood analyses. Genome-wide methylome analysis was performed by the Infinium Human Methylation 450K Bead Chip. The multigene panel analysis revealed the presence of two MMR gene truncating mutations not previously found. Of a total of 15 additional MMR variants identified, five -present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes were found in 12 probands. Methylome analysis detected one constitutional MLH1 epimutation, but no additional differentially methylated regions were identified in LLS compared to LS patients or cancer-free individuals. In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allowed the identification of deleterious MMR mutations as well as new LLS candidate causal genes. Constitutional epimutations in non-LS-associated genes are not responsible for LLS.

Preoperative risk stratification in endometrial cancer (ENDORISK) by a Bayesian network model: A development and validation study.

BACKGROUND: Bayesian networks (BNs) are machine-learning-based computational models that visualize causal relationships and provide insight into the processes underlying disease progression, closely resembling clinical decision-making. Preoperative identification of patients at risk for lymph node metastasis (LNM) is challenging in endometrial cancer, and although several biomarkers are related to LNM, none of them are incorporated in clinical practice. The aim of this study was to develop and externally validate a preoperative BN to predict LNM and outcome in endometrial cancer patients. METHODS AND FINDINGS: Within the European Network for Individualized Treatment of Endometrial Cancer (ENITEC), we performed a retrospective multicenter cohort study including 763 patients, median age 65 years (interquartile range [IQR] 58-71), surgically treated for endometrial cancer between February 1995 and August 2013 at one of the 10 participating European hospitals. A BN was developed using score-based machine learning in addition to expert knowledge. Our main outcome measures were LNM and 5-year disease-specific survival (DSS). Preoperative clinical, histopathological, and molecular biomarkers were included in the network. External validation was performed using 2 prospective study cohorts: the Molecular Markers in Treatment in Endometrial Cancer (MoMaTEC) study cohort, including 446 Norwegian patients, median age 64 years (IQR 59-74), treated between May 2001 and 2010; and the PIpelle Prospective ENDOmetrial carcinoma (PIPENDO) study cohort, including 384 Dutch patients, median age 66 years (IQR 60-73), treated between September 2011 and December 2013. A BN called ENDORISK (preoperative risk stratification in endometrial cancer) was developed including the following predictors: preoperative tumor grade; immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), p53, and L1 cell adhesion molecule (L1CAM); cancer antigen 125 serum level; thrombocyte count; imaging results on lymphadenopathy; and cervical cytology. In the MoMaTEC cohort, the area under the curve (AUC) was 0.82 (95% confidence interval [CI] 0.76-0.88) for LNM and 0.82 (95% CI 0.77-0.87) for 5-year DSS. In the PIPENDO cohort, the AUC for 5-year DSS was 0.84 (95% CI 0.78-0.90). The network was well-calibrated. In the MoMaTEC cohort, 249 patients (55.8%) were classified with <5% risk of LNM, with a false-negative rate of 1.6%. A limitation of the study is the use of imputation to correct for missing predictor variables in the development cohort and the retrospective study design. CONCLUSIONS: In this study, we illustrated how BNs can be used for individualizing clinical decision-making in oncology by incorporating easily accessible and multimodal biomarkers. The network shows the complex interactions underlying the carcinogenetic process of endometrial cancer by its graphical representation. A prospective feasibility study will be needed prior to implementation in the clinic.

Characterisation of the inflammatory response triggered by topical ingenol mebutate 0.05% gel in basal cell carcinoma.

BACKGROUND/OBJECTIVE: Ingenol mebutate gel is approved for actinic keratosis field therapy, but little has been published as a treatment of basal cell carcinoma (BCC). Our objective is to characterise the histopathological changes and the infiltrating cell populations to better understand its mechanism of action. METHODS: Sixteen patients with various BCC subtypes were prospectively evaluated and treated once daily for two consecutive days with ingenol mebutate gel 0.05% under occlusion. Patients were randomised to two arms: the first arm was biopsied between the third and the tenth day after treatment initiation ('early immune response'), and the second arm was biopsied at day 30 after treatment initiation ('late immune response'). The immunopathology was evaluated by immunohistochemistry: anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD56, anti-CD68, anti-Bcl-2, anti-CASP3, anti-FoxP3, anti-GrzB and anti-TIA-1. RESULTS: Ten BCCs were in complete remission after 2 years of follow-up. The early immune response was characterised by a quick recruitment of T lymphocytes, macrophages and natural killer cells. At later time-points, T-regulatory cells and some pro-apoptotic markers were detected. Treatment-related adverse events were described. CONCLUSION: Ingenol mebutate gel produces a transient immuno-inflammatory response and an important necrosis reaction in BCCs. Larger studies will be required to determine the maximum effective tolerated dose of ingenol mebutate gel for BCC.

Intratumour heterogeneity in endometrial serous carcinoma assessed by targeted sequencing and multiplex ligation-dependent probe amplification: a descriptive study.

AIMS: Endometrial serous carcinoma (ESC) represents the most aggressive subtype of endometrial carcinoma (EC). According to The Cancer Genome Atlas (TCGA), ESC exhibits a genomic profile characterised by frequent TP53 mutations and somatic copy-number alterations (SCNA). Several studies have suggested the role of intratumour heterogeneity (ITH) in tumour progression and therapy resistance, highlighting ITH as a challenge for personalised medicine. ITH is described as the co-existence of clonal and subclonal cellular populations within a single tumour. To date, the extent and prevalence of ITH in ESC have not been fully evaluated. The aim of this study was to address ITH analysis in ESC. We performed a descriptive integrated molecular approach using targeted sequencing and multiplex ligation-dependent probe amplification (MLPA) to identify mutations and SCNA patterns, respectively. METHODS AND RESULTS: Eight ESC were examined, selecting three tumour regions per case and their corresponding normal tissue. For targeted sequencing a gene panel of 40 genes based on TCGA and other survey data was performed. For MLPA different probe mixes were used to detect SCNA in 106 genes. Analysis of mutations and SCNA were performed in each sample and comparative analysis of the three tumour regions was also conducted. Targeted sequencing showed that mutations in TP53, PIK3CA and PPP2R1A were ubiquitous in all tumour regions. Moreover, MLPA results demonstrated a high frequency of SCNA, according to the already known presence of genomic instability in ESC. Unlike the homogeneous distribution of somatic mutations, SCNA exhibited ITH affecting targetable genes such as ERBB2. CONCLUSIONS: Our study suggests that somatic gene copy-number alterations are the main source of ITH in ESC.

Poly(adenosine diphosphate ribose) polymerase inhibitors induce autophagy-mediated drug resistance in ovarian cancer cells, xenografts, and patient-derived xenograft models.

BACKGROUND: Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors exhibit promising activity against ovarian cancers, but their efficacy can be limited by acquired drug resistance. This study explores the role of autophagy in regulating the sensitivity of ovarian cancer cells to PARP inhibitors. METHODS: Induction of autophagy was detected by punctate LC3 fluorescence staining, LC3I to LC3II conversion on Western blot analysis, and electron microscopy. Enhanced growth inhibition and apoptosis were observed when PARP inhibitors were used with hydroxychloroquine, chloroquine (CQ), or LYS05 to block the hydrolysis of proteins and lipids in autophagosomes or with small interfering RNA against ATG5 or ATG7 to prevent the formation of autophagosomes. The preclinical efficacy of the combination of CQ and olaparib was evaluated with a patient-derived xenograft (PDX) and the OVCAR8 human ovarian cancer cell line. RESULTS: Four PARP inhibitors (olaparib, niraparib, rucaparib, and talazoparib) induced autophagy in a panel of ovarian cancer cells. Inhibition of autophagy with CQ enhanced the sensitivity of ovarian cancer cells to PARP inhibitors. In vivo, olaparib and CQ produced additive growth inhibition in OVCAR8 xenografts and a PDX. Olaparib inhibited PARP activity, and this led to increased reactive oxygen species (ROS) and an accumulation of γ-H2AX. Inhibition of autophagy also increased ROS and γ-H2AX and enhanced the effect of olaparib on both entities. Treatment with olaparib increased phosphorylation of ATM and PTEN while decreasing the phosphorylation of AKT and mTOR and inducing autophagy. CONCLUSIONS: PARP inhibitor-induced autophagy provides an adaptive mechanism of resistance to PARP inhibitors in cancer cells with wild-type BRCA, and a combination of PARP inhibitors with CQ or other autophagy inhibitors could improve outcomes for patients with ovarian cancer.

Changes of CD68, CD163, and PD-L1 tumor expression during high-dose-rate and pulsed-dose-rate brachytherapy for cervical cancer.

PURPOSE: We hypothesized that radiation doses delivered with high-dose-rate (HDR) and pulsed-dose-rate (PDR) brachytherapy in patients with cervical cancer could trigger immune stimulation by modulating immune cells in the tumor microenvironment. The objective was to determine CD68, CD163, and PD-L1 expression in biopsies from patients with cervical cancer and to compare the effects of HDR vs. PDR brachytherapy on the expression of these proteins. METHODS AND MATERIALS: Nineteen consecutive women (mean age, 55.9 years) with histologically proven cervical cancer scheduled for brachytherapy after standard external beam irradiation therapy combined with platinum-based chemotherapy were included in a prospective study. Core tissue biopsies were obtained before radiochemotherapy (biopsy #1), after completion of radiochemotherapy and before brachytherapy (biopsy #2), and 2 weeks after completion of brachytherapy (biopsy #3). HDR or PDR brachytherapy was delivered according to availability of equipment. CD68, CD163, and PD-L1 immunohistochemical expression was estimated using a quantitative method. Conditional logistic regression models were used to assess the relationship between gene expression and time of biopsy for each brachytherapy technique. RESULTS: In relation to CD68 and CD163, statistically significant relationships between gene expression and biopsy tissue samples were not found in any of the brachytherapy techniques, although there was trend toward downexpression of CD68 and CD163 in biopsies #2 and #3 in the HDR brachytherapy cohort only. There was a significant increase in PD-L1 expression in biopsy #3 also in the HDR cohort as compared with the PDR cohort. CONCLUSIONS: Decreased CD68 and CD163 expression did not reach statistical significance, but this trend may have clinical translational potential. Overexpression of PD-L1 in tissue biopsies taken at 14 days in the HDR brachytherapy cohort may tentatively suggest that this time period would be an adequate interval for blockade of the PD-1/PD-L1 axis.

Addition of IMP3 to L1CAM for discrimination between low- and high-grade endometrial carcinomas: a European Network for Individualised Treatment of Endometrial Cancer collaboration study.

Discrimination between low- and high-grade endometrial carcinomas (ECs) is clinically relevant but can be challenging for pathologists, with moderate interobserver agreement. Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is an oncofoetal protein that is associated with nonendometrioid endometrial carcinomas but has been limited studied in endometrioid carcinomas. The aim of this study is to investigate the diagnostic and prognostic value of IMP3 in the discrimination between low- and high-grade ECs and its added value to L1CAM. IMP3 and L1CAM expression was assessed in tumors from 378 patients treated for EC at 1 of 9 participating European Network for Individualised Treatment of Endometrial Cancer centers. IMP3 was expressed in 24.6% of the tumors. In general, IMP3 was more homogeneously expressed than L1CAM. IMP3 expression was significantly associated with advanced stage, nonendometrioid histology, grade 3 tumors, deep myometrial invasion, lymphovascular space invasion, distant recurrences, overall mortality, and disease-related mortality. Simultaneous absence of IMP3 and L1CAM expression showed the highest accuracy for identifying low-grade carcinomas (area under the curve 0.766), whereas simultaneous expression of IMP3 and L1CAM was strongly associated with high-grade carcinomas (odds ratio 19.7; 95% confidence interval 9.2-42.2). Even within endometrioid carcinomas, this combination remained superior to IMP3 and L1CAM alone (odds ratio 8.6; 95% confidence interval 3.4-21.9). In conclusion, IMP3 has good diagnostic value and together with L1CAM represents the optimal combination of diagnostic markers for discrimination between low- and high-grade ECs compared to IMP3 and L1CAM alone. Because of the homogenous expression of IMP3, this marker might be valuable in preoperative biopsies when compared to the more patchy L1CAM expression.