DNA-barcoded signal amplification for imaging mass cytometry enables sensitive and highly multiplexed tissue imaging.

Imaging mass cytometry (IMC) is a highly multiplexed, antibody-based imaging method that captures heterogeneous spatial protein expression patterns at subcellular resolution. Here we report the extension of IMC to low-abundance markers through incorporation of the DNA-based signal amplification by exchange reaction, immuno-SABER. We applied SABER-IMC to image the tumor immune microenvironment in human melanoma by simultaneous imaging of 18 markers with immuno-SABER and 20 markers without amplification. SABER-IMC enabled the identification of immune cell phenotypic markers, such as T cell co-receptors and their ligands, that are not detectable with IMC.

Lung adenocarcinoma patients with malignant pleural effusions in hot adaptive immunity status have a longer overall survival.

Malignant pleural effusion (MPE) is a common complication of lung adenocarcinoma (LADC) which is associated with a dismal prognosis. We investigated the prognostic role of PD-L1 and other immunomodulators expression in the immune compartment of MPE immune composition. MPE cytologic cell blocks of 83 LADC patients were analysed for the mRNA expression of 770 cancer-immune genes by the NanoString nCounter platform. The expression of relevant immune cell lineage markers was validated by immunohistochemistry (IHC) using quantitative pathology. The mRNA immune profiling identified four MPE patient clusters (C). C1/2 (adaptive+, hot) showed better overall survival (OS) than C3/4 (adaptive-, cold). Additionally, cold immunity profiles (adaptive-), C4 (innate+) were associated with worse OS than C3 (innate-). High PD-L1 expression was linked to the regulation of T cell activation and interferon signalling pathways. Genes of pattern recognition receptor and type I interferon signalling pathways were specifically upregulated in the long-survival (≥90 days) patient group. Moreover, immunomodulators were co-activated and highly expressed in hot adaptive immunity patient clusters, whereas CD274 (PD-L1), TNFRSF9 (4-1BB), VEGFA (VEGF-A) and CD276 (B7-H3) were upregulated in the groups referred as cold. The patient cluster, age and PD-L1 expression were independent prognosticators for LADC MPE patients (p-value < 0.05). Our study sheds light on the variances of immune contexture regarding different PD-L1 expression and survival conditions. It revealed four distinct prognostic patient clusters with specific immune cell components and immunomodulator expression profiles, which, collectively, is supportive for future therapeutic and prognosis for cancer management.

The Tumor Profiler Study: integrated, multi-omic, functional tumor profiling for clinical decision support.

The application and integration of molecular profiling technologies create novel opportunities for personalized medicine. Here, we introduce the Tumor Profiler Study, an observational trial combining a prospective diagnostic approach to assess the relevance of in-depth tumor profiling to support clinical decision-making with an exploratory approach to improve the biological understanding of the disease.

Enhanced prognostic stratification of neoadjuvant treated lung squamous cell carcinoma by computationally-guided tumor regression scoring.

INTRODUCTION: The amount of residual tumor burden after neoadjuvant chemotherapy is an important prognosticator, but for non-small cell lung carcinoma (NSCLC), no official regression scoring system is yet established. Computationally derived histological regression scores could provide unbiased and quantitative readouts to complement the clinical assessment of treatment response. METHODS: Histopathologic tumor regression was microscopically assessed on whole cases in a neoadjuvant chemotherapy-treated cohort (NAC, n = 55 patients) of lung squamous cell carcinomas (LSCC). For each patient, the slide showing the least pathologic regression was selected for subsequent computational analysis and histological features were quantified: percentage of vital tumor cells (cTu.Percentage), total surface covered by vital tumor cells (cTu.Area), area of the largest vital tumor fragment (cTu.Size.max), and total number of vital tumor fragments (cTu.Fragments). A chemo-naïve LSCC cohort (CN, n = 104) was used for reference. For 23 of the 55 patients [18F]-Fluorodeoxyglucose (FDG) PET/CT measurements of maximum standard uptake value (SUVmax), background subtracted lesion activity (BSL) and background subtracted volume (BSV) were correlated with pathologic regression. Survival analysis was carried out using Cox regression and receiver operating characteristic (ROC) curve analysis using a 3-years cutoff. RESULTS: All computational regression parameters significantly correlated with relative changes of BSV FDG PET/CT values after neoadjuvant chemotherapy. ROC curve analysis of histological parameters of NAC patients showed that cTu.Percentage was the most accurate prognosticator of overall survival (ROC curve AUC = 0.77, p-value = 0.001, Cox regression HR = 3.6, p = 0.001, variable cutoff < = 30 %). CONCLUSIONS: This study demonstrates the prognostic relevance of computer-derived histopathologic scores. Additionally, the analysis carried out on slides displaying the least pathologic regression correlated with overall pathologic response and PET/CT values. This might improve the objective histopathologic assessment of tumor response in neoadjuvant setting.

In vitro cell culture of patient derived malignant pleural and peritoneal effusions for personalised drug screening.

BACKGROUND: Malignant serous effusion (MSE) denotes a manifestation of metastatic disease with typical high concentrations of both cancer and immune cells, making them an ideal resource for in vitro cytologic studies. Hence, the aim of the study was to investigate the features of 2D and 3D MSE culture systems as well as their feasibilities for in vitro drug screening. METHODS: Pleural and peritoneal effusions from 8 patients were collected and processed for 2D monolayer and 3D hanging drop cell culture into GravityPLUS™ plates. Representative markers for cell components, proliferation rate and tumour classification were investigated by immunohistochemistry, followed by absolute quantification using a digitalised image analysis approach. Further, we implemented another 3D cell culture model based on a low attachment method for in vitro drug sensitivity testing of carboplatin, pemetrexed and pembrolizumab for 5 patients. RESULTS: Monolayer cell culture was favourable for the growth of mesothelial cells, while hanging drop culture in GravityPLUS™ plates showed better ability for preserving cancer cells, inducing positive diagnostic markers expression and restraining the growth of mesothelial cells. For in vitro drug testing, MSE from five patients presented various drug sensitivities, and one case showed strong response to PD-1 checkpoint inhibition (pembrolizumab). For some patients, the application of combinatorial drugs had better therapeutic responses compared to monotherapy. CONCLUSIONS: Digitalised quantification of data offers a better understanding of different MSE culture models. More importantly, the proposed platforms are practical and amenable for performing in vitro chemo-/immunotherapeutic drug testing by using routine cytologic MSE in a personalised manner. Next to cell blocks, our work demonstrates the prognostic and predictive value of cytologic effusion samples.

Prognostic Immune Cell Profiling of Malignant Pleural Effusion Patients by Computerized Immunohistochemical and Transcriptional Analysis.

Malignant pleural effusion (MPE) is a severe condition of advanced tumors without effective therapy. We used digitalized immunohistochemical and transcriptional approaches to investigate the prognostic influence of immune cells and expression variance of associated immunomodulatory molecules in MPE. Cytology tissue microarrays were constructed from MPE cell blocks of 155 patients with five tumor entities. Immune cells lineage markers were quantified by computational cytopathology on immunohistochemistry. mRNA expression analysis of nine lineage markers and 17 immunomodulators was performed by NanoString. Immunohistochemically quantified high B cells to leukocytes ratio (hazard ratio (HR) = 0.70, p-value = 0.043) and low neutrophils to leukocytes ratio (HR = 1.78, p-value = 0.003) were favorable prognosticators for overall survival independent of tumor entity. Correspondingly, patients with high B cells but low neutrophils gene expression signature showed longer median overall survival of 500 days (HR = 2.29, p-value = 0.009). Regarding targetable molecule expressions, lung adenocarcinomas were characterized by high PD-L1, but mesothelioma by high LAG-3. Ovarian carcinoma was least immunogenic. Independent of tumor entity, the condition of the immune system in MPE liquids is able to provide additional prognostic cytologic information. Combined analysis of lineage specific markers and related immunomodulators may direct immune-based therapeutic decisions.

Computer-Based Intensity Measurement Assists Pathologists in Scoring Phosphatase and Tensin Homolog Immunohistochemistry - Clinical Associations in NSCLC Patients of the European Thoracic Oncology Platform Lungscape Cohort.

INTRODUCTION: Phosphatase and tensin homolog (PTEN) loss is frequently observed in NSCLC and associated with both phosphoinositide 3-kinase activation and tumoral immunosuppression. PTEN immunohistochemistry is a valuable readout, but lacks standardized staining protocol and cutoff value. METHODS: After an external quality assessment using SP218, 138G6 and 6H2.1 anti-PTEN antibodies, scored on webbook and tissue microarray, the European Thoracic Oncology Platform cohort samples (n = 2245 NSCLC patients, 8980 tissue microarray cores) were stained with SP218. All cores were H-scored by pathologists and by computerized pixel-based intensity measurements calibrated by pathologists. RESULTS: All three antibodies differentiated six PTEN+ versus six PTEN- cases on external quality assessment. For 138G6 and SP218, high sensitivity and specificity was found for all H-score threshold values including prospectively defined 0, calculated 8 (pathologists), and calculated 5 (computer). High concordance among pathologists in setting computer-based intensities and between pathologists and computer in H-scoring was observed. Because of over-integration of the human eye, pixel-based computer H-scores were overall 54% lower. For all cutoff values, PTEN- was associated with smoking history, squamous cell histology, and higher tumor stage (p < 0.001). In adenocarcinomas, PTEN- was associated with poor survival. CONCLUSION: Calibration of immunoreactivity intensities by pathologists following computerized H-score measurements has the potential to improve reproducibility and homogeneity of biomarker detection regarding epitope validation in multicenter studies.

A Quantitative Pathology Approach to Analyze the Development of Human Cancer-Associated Tertiary Lymphoid Structures.

Tertiary lymphoid structures (TLS) develop in the human tumor microenvironment and correlate with prolonged survival in most cancer types. We recently demonstrated that TLS development follows sequential maturation stages and culminates in the generation of a germinal center (GC) reaction. This maturation process is crucial for the prognostic relevance of TLS in lung and colorectal cancer patients.The mechanisms underlying TLS development in various inflammatory conditions or their functional relevance in tumor immunity are not fully understood. Investigating which cell types and soluble mediators orchestrate lymphoid neogenesis in human tissues requires a method that allows simultaneous detection of multiple markers.Here, we describe a quantitative pathology approach to identify and quantify different TLS maturation stages in combination with other parameters. This approach consists of seven-color immunofluorescence protocol using tyramide signal amplification combined with multispectral microscopy and quantitative data acquisition from histological images.

Computationally-Guided Development of a Stromal Inflammation Histologic Biomarker in Lung Squamous Cell Carcinoma.

The goal of this study is to use computational pathology to help guide the development of human-based prognostic H&E biomarker(s) suitable for research and potential clinical use in lung squamous cell carcinoma (SCC). We started with high-throughput computational image analysis with tissue microarrays (TMAs) to screen for histologic features associated with patient overall survival, and found that features related to stromal inflammation were the most strongly prognostic. Based on this, we developed an H&E stromal inflammation (SI) score. The prognostic value of the SI score was validated by two blinded human observers on two large cohorts from a single institution. The SI score was found to be reproducible on TMAs (Spearman rho = 0.88 between the two observers), and highly prognostic (e.g. hazard ratio = 0.32; 95% confidence interval: 0.19-0.54; p-value = 2.5 × 10-5 in multivariate analyses), particularly in comparison to established histologic biomarkers. Guided by downstream molecular/biomarker correlation studies starting with TCGA cases, we investigated the hypothesis that epithelial PD-L1 expression modified the prognostic value of SI. Our research demonstrates that computational pathology can be an efficient hypothesis generator for human pathology research, and support the histologic evaluation of SI as a prognostic biomarker in lung SCCs.

Germinal Centers Determine the Prognostic Relevance of Tertiary Lymphoid Structures and Are Impaired by Corticosteroids in Lung Squamous Cell Carcinoma.

In solid tumors, the presence of lymph node-like structures called tertiary lymphoid structures (TLS) is associated with improved patient survival. However, little is known about how TLS develop in cancer, how their function affects survival, and whether they are affected by cancer therapy. In this study, we used multispectral microscopy, quantitative pathology, and gene expression profiling to analyze TLS formation in human lung squamous cell carcinoma (LSCC) and in an experimental model of lung TLS induction. We identified a niche of CXCL13+ perivascular and CXCL12+LTB+ and PD-L1+ epithelial cells supporting TLS formation. We also characterized sequential stages of TLS maturation in LSCC culminating in the formation of germinal centers (GC). In untreated patients, TLS density was the strongest independent prognostic marker. Furthermore, TLS density correlated with GC formation and expression of adaptive immune response-related genes. In patients treated with neoadjuvant chemotherapy, TLS density was similar, but GC formation was impaired and the prognostic value of TLS density was lost. Corticosteroids are coadministered with chemotherapy to manage side effects in LSCC patients, so we evaluated whether they impaired TLS development independently of chemotherapy. TLS density and GC formation were each reduced in chemotherapy-naïve LSCC patients treated with corticosteroids before surgery, compared with untreated patients, a finding that we confirmed in the experimental model of lung TLS induction. Overall, our results highlight the importance of GC formation in TLS during tumor development and treatment.Significance: Corticosteroid treatment during chemotherapy negatively affects the development of tertiary lymphoid structures and abrogates their prognostic value in patients with lung cancer. Cancer Res; 78(5); 1308-20. ©2018 AACR.

Morphoproteomic Characterization of Lung Squamous Cell Carcinoma Fragmentation, a Histological Marker of Increased Tumor Invasiveness.

Accurate stratification of tumors is imperative for adequate cancer management. In addition to staging, morphologic subtyping allows stratification of patients into additional prognostic groups. In this study, we used an image-based computational method on pan-cytokeratin IHC stainings to quantify tumor fragmentation (TF), a measure of tumor invasiveness of lung squamous cell carcinoma (LSCC). In two independent clinical cohorts from tissue microarrays (TMA: n = 208 patients) and whole sections (WS: n = 99 patients), TF was associated with poor prognosis and increased risk of blood vessel infiltration. A third cohort from The Cancer Genome Atlas (TCGA: n = 335 patients) confirmed the poor prognostic value of TF using a similar human-based score on hematoxylin-eosin staining. Integration of RNA-seq data from TCGA and LC-MS/MS proteomics from WS revealed an upregulation of extracellular matrix remodeling and focal adhesion processes in tumors with high TF, supporting their increased invasive potential. This proposed histologic parameter is an independent and unfavorable prognostic marker that could be established as a new grading parameter for LSCC. Cancer Res; 77(10); 2585-93. ©2017 AACR.

Non-internalizing antibody-drug conjugates display potent anti-cancer activity upon proteolytic release of monomethyl auristatin E in the subendothelial extracellular matrix.

Antibody-drug conjugates (ADCs) represent a promising class of biopharmaceuticals with the potential to localize at the tumor site and improve the therapeutic index of cytotoxic drugs. While it is generally believed that ADCs need to be internalized into tumor cells in order to display optimal therapeutic activity, it has recently been shown that non-internalizing antibodies can efficiently liberate disulfide-linked drugs at the extracellular tumor site, leading to potent anti-cancer activity in preclinical animal models. Here, we show that engineered variants of the F16 antibody, specific to a splice isoform of tenascin-C, selectively localize to the subendothelial tumor extracellular matrix in three mouse models of human cancer (U87, A431, MDA-MB-231). A site-specific coupling of F16 in IgG format with a monomethyl auristatin E (MMAE) derivative, featuring a valine-citrulline dipeptide linker equipped with a self-immolative spacer, yielded an ADC product, which cured tumor-bearing mice at a dose of 7 mg/Kg. The observation of an efficient extracellular proteolytic cleavage of the valine-citrulline linker was surprising, as it has generally been assumed that this peptidic structure would be selectively cleaved by cathepsin B in intracellular compartments. The products described in this article may be useful for the treatment of human malignancies, as their cognate antigen is strongly expressed in the majority of human solid tumors, lymphomas and aggressive leukemias, while being virtually undetectable in most normal adult tissues.

18F-FDG PET/CT of Non-Small Cell Lung Carcinoma Under Neoadjuvant Chemotherapy: Background-Based Adaptive-Volume Metrics Outperform TLG and MTV in Predicting Histopathologic Response.

UNLABELLED: Assessment of tumor response after chemotherapy using (18)F-FDG PET metrics is gaining acceptance. Several studies have suggested that the parameters metabolically active tumor volume (MTV) and total lesion glycolysis (TLG) are superior to SUVmax for measuring tumor burden. However, the measurement of MTV and TLG is still controversial; the most common method uses an absolute threshold of 42% of SUVmax Recently, we implemented a background-adaptive method to determine the background-subtracted lesion activity (BSL) and the background-subtracted volume (BSV). In this study, we investigated the correlation between such PET metrics and histopathologic response in non-small cell lung carcinoma (NSCLC). METHODS: Forty-four NSCLC patients were retrospectively identified. Their PET/CT data on both types of scan before and after neoadjuvant chemotherapy were analyzed regarding SUVmax, MTV, TLG, BSL, and BSV, as well as the relative changes in these parameters. The tumor regression score as an indicator of histopathologic response was scored on hematoxylin- and eosin-stained sections of the surgical specimens using a 4-tiered scale (scores 1-4). The correlation between score and the absolute and relative PET metrics after chemotherapy was analyzed using Spearman rank correlation tests. RESULTS: Tumors that demonstrated a good response after neoadjuvant chemotherapy had significantly lower (18)F-FDG activity than nonresponding tumors (scores 3 and 4: SUVmax, 4.2 [range, 1.8-7.9] vs. scores 1 and 2: SUVmax, 8.1 [range, 1.4-40.4]; P = 0.001). The same was found for change in SUVmax and score (P = 0.001). PET volume metrics based on a 42% fixed threshold for SUVmax did not correlate with score (TLG, P = 0.505; MTV, P = 0.386). However, both of the background activity-based PET volume metrics-BSL and BSV-significantly correlated with score (P < 0.001 each). CONCLUSION: PET volume metrics based on background-adaptive methods correlate better with histopathologic tumor regression score in NSCLC patients under neoadjuvant chemotherapy than algorithms and methods using a fixed threshold (42% SUVmax).

[Fibroepithelial polyp of the ureter. Report of one case]. / Pólipo ureteral. Presentación de un caso.

OBJECTIVES: To report the rare case of a patient with a ureteral polyp. METHODS: We describe the case of a 55-year-old female patient receiving care at the Celia Sanchez Manduley University Hospital in Manzanillo, Cuba, who was fortuitously diagnosed of a fibroepithelial polyp of the right ureter during the work up and treatment of an ovarian tumor. RESULTS: This case is the first of its kind in this hospital after 22 years, which confirms the rarity of ureteral tumors, specifically those of benign etiology. The absence of symptoms, specifically hematuria and pain, does not correspond to the reviewed articles. The chosen treatment was exeresis of the polyp at its base and frozen biopsy, followed by re-establishment of the urinary passage, as various authors recommend. Currently the endoscopical approach is recommended for its multiple advantages. CONCLUSIONS: We conclude that this disease is very rare, may have a symptomatic course and the treatment of choice is surgery with very good results.