Independent expression of circulating and tissue levels of PD-L1: correlation of clusters with tumor metabolism and outcome in patients with non-small cell lung cancer.

PURPOSE: To evaluate the clinical-pathological and prognostic significance of the circulating PD-L1 level in patients with surgically treated NSCLC, by combining data for PD-L1 expression with other immune-related markers and tumor metabolism. METHODS: Overall, 40 patients with resected NSCLC (stage Ia-IIIa) who had preoperative blood storage and underwent staging PET/CT were enrolled for the study. In all cases, we determined plasma levels of PD-L1 (pg/ml), immune-reactive areas (IRA %) covered by CD3, CD68, CD20, CD8, PD-1, and PD-L1 in the tumor specimen, and metabolic parameters on PET, i.e., SUVmax, SUVpeak, metabolic tumor volume (MTV), and total lesion glycolysis (TLG). Variables were statistically analyzed to establish their association with disease-free survival (DFS). RESULTS: The circulating levels of PD-L1 in the bloodstream could be determined in 38/40 (95%) samples. The mean and median expression levels were 34.86 pg/ml and 24.83 pg/ml, respectively. We did not find any statistically significant correlation between circulating PD-L1 and tissue expression of PD-L1/PD-1. Some mild degree of positive correlation was determined between tissue PD-L1 and SUVmax (ρ = 0.390; p = 0.0148). Hierarchical clustering combining circulating, tissue, and metabolic parameters identified clusters with high metabolic tumor burden or high expression of plasma PD-L1 levels (Z score ≥ 2) as having a poor DFS (p = 0.033). The multivariate analysis detected stage and metabolism (i.e., SUVmax and SUVpeak) as independent prognostic factors for DFS. CONCLUSION: Plasma levels of PD-L1 are independent of the expression of PD-1/PD-L1 in NSCLC tumor tissue and, when combined with other clinical-pathological parameters, allow for the identification of clusters with different outcomes.

Correlation of metabolic information on FDG-PET with tissue expression of immune markers in patients with non-small cell lung cancer (NSCLC) who are candidates for upfront surgery.

PURPOSE: Eliciting antitumor T-cell response by targeting the PD-1/PD-L1 axis with checkpoint inhibitors has emerged as a novel therapeutic strategy in non-small cell lung cancer (NSCLC). The identification of predictors for sensitivity or resistance to these agents is, therefore, needed. Herein, we investigate the correlation of metabolic information on FDG-PET with tissue expression of immune-checkpoints and other markers of tumor-related immunity in resected NSCLC patients. MATERIALS AND METHODS: All patients referred to our institution for upfront surgical resection of NSCLC, who were investigated with FDG-PET prior to surgery, were consecutively included in the study. From January 2010 to May 2014, 55 patients (stage IA-IIIB; M:F = 42:13; mean age 68.9 years) were investigated. Sampled surgical tumor specimens were analyzed by immunohistochemistry (IHC) for CD68-TAMs (tumor-associated macrophages), CD8-TILs (tumor infiltrating lymphocytes), PD-1-TILs, and PD-L1 tumor expression. Immunoreactivity was evaluated, and scores were compared with imaging findings. FDG-PET images were analyzed to define semi-quantitative parameters: SUVmax and SUVmean. Metabolic information on FDG-PET was correlated with tissue markers expression and disease-free survival (DFS) considering a median follow-up of 16.2 months. RESULTS: Thirty-six adenocarcinomas (ADC), 18 squamous cell carcinomas (SCC), and one sarcomatoid carcinoma were analyzed. All tumors resulted positive at FDG-PET: median SUVmax 11.3 (range: 2.3-32.5) and SUVmean 6.4 (range: 1.5-13) both resulted significantly higher in SCC compared to other NSCLC histotypes (p = 0.007 and 0.048, respectively). IHC demonstrated a median immunoreactive surface covered by CD68-TAMs of 5.41 % (range: 0.84-14.01 %), CD8-TILs of 2.9 % (range: 0.11-11.92 %), PD-1 of 0.65 % (range: 0.02-5.87 %), and PD-L1 of 0.7 % (range: 0.03-10.29 %). We found a statistically significant correlation between SUVmax and SUVmean with the expression of CD8 TILs (rho = 0.31; p = 0.027) and PD-1 (rho = 0.33; p = 0.017 and rho = 0.36; p = 0.009, respectively). The other tissue markers correlated as follows: CD8 TILs and PD-1 (rho = 0.45; p = 0.001), CD8 TILs and PD-L1 (rho = 0.41; p = 0.003), CD68-TAMs and PD-L1 (rho = 0.30; p = 0.027), PD-1 and PD-L1 (rho = 0.26; p = 0.059). With respect to patients' outcome, SUVmax, SUVmean, and disease stage showed a statistically significant correlation with DFS (p = 0.002, 0.004, and <0.001, respectively). CONCLUSIONS: The present study shows a direct association between metabolic parameters on FDG-PET and the expression of tumor-related immunity markers, suggesting a potential role for FDG-PET to characterize the tumor microenvironment and select NSCLC patients candidate to checkpoint inhibitors.

The Smokers Health Multiple ACtions (SMAC-1) Trial: Study Design and Results of the Baseline Round.

BACKGROUND: Lung cancer screening with low-dose helical computed tomography (LDCT) reduces mortality in high-risk subjects. Cigarette smoking is linked to up to 90% of lung cancer deaths. Even more so, it is a key risk factor for many other cancers and cardiovascular and pulmonary diseases. The Smokers health Multiple ACtions (SMAC-1) trial aimed to demonstrate the feasibility and effectiveness of an integrated program based on the early detection of smoking-related thoraco-cardiovascular diseases in high-risk subjects, combined with primary prevention. A new multi-component screening design was utilized to strengthen the framework on conventional lung cancer screening programs. We report here the study design and the results from our baseline round, focusing on oncological findings. METHODS: High-risk subjects were defined as being >55 years of age and active smokers or formers who had quit within 15 years (>30 pack/y). A PLCOm2012 threshold >2% was chosen. Subject outreach was streamlined through media campaign and general practitioners' engagement. Eligible subjects, upon written informed consent, underwent a psychology consultation, blood sample collection, self-evaluation questionnaire, spirometry, and LDCT scan. Blood samples were analyzed for pentraxin-3 protein levels, interleukins, microRNA, and circulating tumor cells. Cardiovascular risk assessment and coronary artery calcium (CAC) scoring were performed. Direct and indirect costs were analyzed focusing on the incremental cost-effectiveness ratio per quality-adjusted life years gained in different scenarios. Personalized screening time-intervals were determined using the "Maisonneuve risk re-calculation model", and a threshold <0.6% was chosen for the biennial round. RESULTS: In total, 3228 subjects were willing to be enrolled. Out of 1654 eligible subjects, 1112 participated. The mean age was 64 years (M/F 62/38%), with a mean PLCOm2012 of 5.6%. Former and active smokers represented 23% and 77% of the subjects, respectively. At least one nodule was identified in 348 subjects. LDCTs showed no clinically significant findings in 762 subjects (69%); thus, they were referred for annual/biennial LDCTs based on the Maisonneuve risk (mean value = 0.44%). Lung nodule active surveillance was indicated for 122 subjects (11%). Forty-four subjects with baseline suspicious nodules underwent a PET-FDG and twenty-seven a CT-guided lung biopsy. Finally, a total of 32 cancers were diagnosed, of which 30 were lung cancers (2.7%) and 2 were extrapulmonary cancers (malignant pleural mesothelioma and thymoma). Finally, 25 subjects underwent lung surgery (2.25%). Importantly, there were zero false positives and two false negatives with CT-guided biopsy, of which the patients were operated on with no stage shift. The final pathology included lung adenocarcinomas (69%), squamous cell carcinomas (10%), and others (21%). Pathological staging showed 14 stage I (47%) and 16 stage II-IV (53%) cancers. CONCLUSIONS: LDCTs continue to confirm their efficacy in safely detecting early-stage lung cancer in high-risk subjects, with a negligible risk of false-positive results. Re-calculating the risk of developing lung cancer after baseline LDCTs with the Maisonneuve model allows us to optimize time intervals to subsequent screening. The Smokers health Multiple ACtions (SMAC-1) trial offers solid support for policy assessments by policymakers. We trust that this will help in developing guidelines for the large-scale implementation of lung cancer screening, paving the way for better outcomes for lung cancer patients.

Important functional role of the protein osteopontin in the progression of malignant pleural mesothelioma.

Background: Malignant Pleural Mesothelioma (MPM) is an aggressive cancer of the mesothelial lining associated with exposure to airborne non-degradable asbestos fibers. Its poor response to currently available treatments prompted us to explore the biological mechanisms involved in its progression. MPM is characterized by chronic non-resolving inflammation; in this study we investigated which inflammatory mediators are mostly expressed in biological tumor samples from MPM patients, with a focus on inflammatory cytokines, chemokines and matrix components. Methods: Expression and quantification of Osteopontin (OPN) was detected in tumor and plasma samples of MPM patients by mRNA, immunohistochemistry and ELISA. The functional role of OPN was investigated in mouse MPM cell lines in vivo using an orthotopic syngeneic mouse model. Results: In patients with MPM, the protein OPN was significantly more expressed in tumors than in normal pleural tissues and predominantly produced by mesothelioma cells; plasma levels were elevated in patients and associated with poor prognosis. However, modulation of OPN levels was not significantly different in a series of 18 MPM patients receiving immunotherapy with durvalumab alone or with pembrolizumab in combination with chemotherapy, some of whom achieved a partial clinical response. Two established murine mesothelioma cell lines: AB1 and AB22 of sarcomatoid and epithelioid histology, respectively, spontaneously produced high levels of OPN. Silencing of the OPN gene (Spp1) dramatically inhibited tumor growth in vivo in an orthotopic model, indicating that OPN has an important promoting role in the proliferation of MPM cells. Treatment of mice with anti-CD44 mAb, blocking a major OPN receptor, significantly reduced tumor growth in vivo. Conclusion: These results demonstrate that OPN is an endogenous growth factor for mesothelial cells and inhibition of its signaling may be helpful to restrain tumor progression in vivo. These findings have translational potential to improve the therapeutic response of human MPM.

Muscle and Muscle-like Autoantigen Expression in Myasthenia Gravis Thymus: Possible Molecular Hint for Autosensitization.

The thymus is widely recognized as an immunological niche where autoimmunity against the acetylcholine receptor (AChR) develops in myasthenia gravis (MG) patients, who mostly present thymic hyperplasia and thymoma. Thymoma-associated MG is frequently characterized by autoantibodies to the muscular ryanodine receptor 1 (RYR1) and titin (TTN), along with anti-AChR antibodies. By real-time PCR, we analyzed muscle-CHRNA1, RYR1, and TTN-and muscle-like-NEFM, RYR3 and HSP60-autoantigen gene expression in MG thymuses with hyperplasia and thymoma, normal thymuses and non-MG thymomas, to check for molecular changes potentially leading to an altered antigen presentation and autoreactivity. We found that CHRNA1 (AChR-α subunit) and AIRE (autoimmune regulator) genes were expressed at lower levels in hyperplastic and thymoma MG compared to the control thymuses, and that the RYR1 and TTN levels were decreased in MG versus the non-MG thymomas. Genes encoding autoantigens that share epitopes with AChR-α (NEFM and HSP60), RYR1 (neuronal RYR3), and TTN (NEFM) were up-regulated in thymomas versus hyperplastic and control thymuses, with distinct molecular patterns across the thymoma histotypes that could be relevant for autoimmunity development. Our findings support the idea that altered muscle autoantigen expression, related with hyperplastic and neoplastic changes, may favor autosensitization in the MG thymus, and that molecular mimicry involving tumor-related muscle-like proteins may be a mechanism that makes thymoma prone to developing MG.

Oncogenic KRAS-Induced Protein Signature in the Tumor Secretome Identifies Laminin-C2 and Pentraxin-3 as Useful Biomarkers for the Early Diagnosis of Pancreatic Cancer.

KRAS mutations characterize pancreatic cell transformation from the earliest stages of carcinogenesis, and are present in >95% of pancreatic ductal adenocarcinoma (PDAC) cases. In search of novel biomarkers for the early diagnosis of PDAC, we identified the proteins secreted by the normal human pancreatic cell line (HPDE) recently transformed by inducing the overexpression of the KRASG12V oncogene. We report a proteomic signature of KRAS-induced secreted proteins, which was confirmed in surgical tumor samples from resected PDAC patients. The putative diagnostic performance of three candidates, Laminin-C2 (LAMC2), Tenascin-C (TNC) and Pentraxin-3 (PTX3), was investigated by ELISA quantification in two cohorts of PDAC patients (n = 200) eligible for surgery. Circulating levels of LAMC2, TNC and PTX3 were significantly higher in PDAC patients compared to the healthy individuals (p < 0.0001). The Receiver Operating Characteristics (ROC) curve showed good sensitivity (1) and specificity (0.63 and 0.85) for LAMC2 and PTX3, respectively, but not for TNC, and patients with high levels of LAMC2 had significantly shorter overall survival (p = 0.0007). High levels of LAMC2 and PTX3 were detected at early stages (I−IIB) and in CA19-9-low PDAC patients. In conclusion, pancreatic tumors release LAMC2 and PTX3, which can be quantified in the systemic circulation, and may be useful in selecting patients for further diagnostic imaging.

Mutual exclusion of sensory bristles and tendons on the notum of dipteran flies.

BACKGROUND: Genes of the achaete-scute complex encode transcription factors whose activity regulates the development of neural cells. The spatially restricted expression of achaete-scute on the mesonotum of higher flies governs the development and positioning of the large sensory bristles. On the scutum the bristles are arranged into conserved patterns, based on an ancestral arrangement of four longitudinal rows. This pattern appears to date back to the origin of cyclorraphous flies about 100-140 million years ago. The origin of the four-row bauplan, which is independent of body size, and the reasons for its conservation, are not known. RESULTS: We report that tendons for attachment of the indirect flight muscles are invariably located between the bristle rows of the scutum throughout the Diptera. Tendon development depends on the activity of a transcription factor encoded by the gene stripe. In Drosophila, stripe and achaete-scute have separate expression domains, leading to spatial segregation of tendon precursors and bristle precursors. Furthermore the products of these genes act antagonistically: ectopic sr expression prevents bristle development and ectopic sc expression prevents normal muscle attachment. The product of stripe acts downstream of Achaete-Scute and interferes with the development of bristle precursors. CONCLUSIONS: The pattern of flight muscles has changed little throughout the Diptera and we argue that the sites of muscle attachment may have constrained the positioning of bristles during the course of evolution. This could account for the pattern of four bristle rows on the scutum.

Gene duplication at the achaete-scute complex and morphological complexity of the peripheral nervous system in Diptera.

The number of achaete-scute genes increased during insect evolution, particularly in the Diptera lineage. Sequence comparison indicates that the four achaete-scute genes of Drosophila result from three independent duplication events. After duplication, the new genes acquired individual expression patterns but, in Drosophila, their products can compensate for one another, which raises the question: why retain all four genes? The complexity of the spatial expression of these genes on the notum increased in the lineage leading to the higher Diptera, allowing the development of stereotyped bristle patterns. This probably coincided in time with gene duplication events, raising the possibility that an increase in gene copy number might have provided the flexibility necessary for more complex transcriptional regulation.

Parallel Activin and BMP signaling coordinates R7/R8 photoreceptor subtype pairing in the stochastic Drosophila retina.

Drosophila color vision is achieved by comparing outputs from two types of color-sensitive photoreceptors, R7 and R8. Ommatidia (unit eyes) are classified into two subtypes, known as 'pale' or 'yellow', depending on Rhodopsin expression in R7 and R8. Subtype specification is controlled by a stochastic decision in R7 and instructed to the underlying R8. We find that the Activin receptor Baboon is required in R8 to receive non-redundant signaling from the three Activin ligands, activating the transcription factor dSmad2. Concomitantly, two BMP ligands activate their receptor, Thickveins, and the transcriptional effector, Mad. The Amon TGFß processing factor appears to regulate components of the TGFß pathway specifically in pale R7. Mad and dSmad2 cooperate to modulate the Hippo pathway kinase Warts and the growth regulator Melted; two opposing factors of a bi-stable loop regulating R8 Rhodopsin expression. Therefore, TGFß and growth pathways interact in postmitotic cells to precisely coordinate cell-specific output.

How to pattern an epithelium: lessons from achaete-scute regulation on the notum of Drosophila.

The notum of Drosophila is a good model system for the study of two-dimensional pattern formation. Attention has mainly focused on the regulation of the spatial expression of the genes of the achaete-scute complex (AS-C) that results in a stereotyped bristle pattern. Expression of AS-C genes has traditionally been viewed as a consequence of the activity of a group of factors that constitute a prepattern [Stern, 1954. Am. Sci. 42, 213]. The prepattern is thought to be composed of a mosaic of transcription factors that act in combination, through discrete cis-regulatory sequences, to activate expression of genes of the AS-C in small clusters of cells at the sites of each future bristle. Recent results challenge this view and suggest a hierarchy of activity amongst prepattern genes. It is suggested that in the medial notum, the selector-like gene pannier regulates the entire pattern, and is the only factor to directly activate AS-C genes. Other factors may play subsidiary roles. On the lateral notum genes of the iroquois complex appear to regulate the lateral pattern. Regulation of pannier and iroquois depends upon the signalling molecule Decapentaplegic. The majority of genes are expressed in either longitudinal or transverse domains on the notum and we discuss the possibility that pattern formation may rely on these two axial coordinates. We also discuss preliminary results suggesting that prepattern factors also regulate genes required for other, little studied, aspects of notal morphology, such as the muscle attachment sites and pigment distribution. Thus there may be a common prepattern for the entire structure.

Opposite feedbacks in the Hippo pathway for growth control and neural fate.

Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and oncoprotein YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase LATS/Warts (Wts) and growth regulator Melted generates mutually exclusive photoreceptor subtypes. Here, we show that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network-including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module-that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution.

Minimum biobanking requirements: issues in a comprehensive cancer center biobank.

Cancer biobanks, when located within a comprehensive cancer center, are characterized by management and organizational peculiarities mainly related to the multidisciplinary information available of such specialized centers and the continuous collection stream of quality-assessed biospecimens. The present study summarizes the main characteristics of comprehensive cancer center biobanks and, more in detail, procedures addressed in order to maintain full control over interlacement issues that occur at every level, from patient enrolment eligibility and consenting to dissemination and utilization of specimens and associated data. Dedicated personnel, appropriate storage facilities, as well as ethical, legal, and technical requirements are among the most relevant aspects strongly conditioning the quality of these structures. Because of its location and the need to be directly connected with clinical units, such as pathology, oncology, surgery, etc., ad hoc information technology tools are crucial to support all aspects of biorepository operations, including (but not limited to) patient enrolment and consent; biospecimen collection, processing, storage, and distribution; quality assurance and quality control; collection of patient data; validation documentation; and management reporting functions.

NF-kappaB/Rel-mediated regulation of the neural fate in Drosophila.

Two distinct roles are described for Dorsal, Dif and Relish, the three NF-kappaB/Rel proteins of Drosophila, in the development of the peripheral nervous system. First, these factors regulate transcription of scute during the singling out of sensory organ precursors from clusters of cells expressing the proneural genes achaete and scute. This effect is possibly mediated through binding sites for NF-kappaB/Rel proteins in a regulatory module of the scute gene required for maintenance of scute expression in precursors as well as repression in cells surrounding precursors. Second, genetic evidence suggests that the receptor Toll-8, Relish, Dif and Dorsal, and the caspase Dredd pathway are active over the entire imaginal disc epithelium, but Toll-8 expression is excluded from sensory organ precursors. Relish promotes rapid turnover of transcripts of the target genes scute and asense through an indirect, post-transcriptional mechanism. We propose that this buffering of gene expression levels serves to keep the neuro-epithelium constantly poised for neurogenesis.

Flipping coins in the fly retina.

Color vision in Drosophila melanogaster relies on the presence of two different subtypes of ommatidia: the "green" and "blue." These two classes are distributed randomly throughout the retina. The decision of a given ommatidium to take on the "green" or "blue" fate seems to be based on a stochastic mechanism. Here we compare the stochastic choice of photoreceptors in the fly retina with other known examples of random choices in both sensory and other systems.

The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors.

Color vision in Drosophila relies on the comparison between two color-sensitive photoreceptors, R7 and R8. Two types of ommatidia in which R7 and R8 contain different rhodopsins are distributed stochastically in the retina and appear to discriminate short (p-subset) or long wavelengths (y-subset). The choice between p and y fates is made in R7, which then instructs R8 to follow the corresponding fate, thus leading to a tight coupling between rhodopsins expressed in R7 and R8. Here, we show that warts, encoding large tumor suppressor (Lats) and melted encoding a PH-domain protein, play opposite roles in defining the yR 8 or pR8 fates. By interacting antagonistically at the transcriptional level, they form a bistable loop that insures a robust commitment of R8 to a single fate, without allowing ambiguity. This represents an unexpected postmitotic role for genes controlling cell proliferation (warts and its partner hippo and salvador) and cell growth (melted).

scute expression in Calliphora vicina reveals an ancestral pattern of longitudinal stripes on the thorax of higher Diptera.

In Drosophila the stereotyped arrangement of sensory bristles on the notum is determined by the tightly regulated control of transcription of the achaete-scute (ac-sc) genes which are expressed in small proneural clusters of cells at the sites of each future bristle. Expression relies on a series of discrete cis-regulatory elements present in the ac-sc gene complex that are the target of the transcriptional activators pannier (pnr) and the genes of the iroquois complex. Stereotyped bristle patterns are common among species of acalyptrate Schizophora such as Drosophila, and are thought to have derived from an ancestral pattern of four longitudinal rows extending the length of the scutum, through secondary loss of bristles. To investigate evolutionary changes in bristle patterns and ac-sc regulation by pnr, we have isolated homologues of these genes from Calliphora vicina, a species of calyptrate Schizophora separated from Drosophila by at least 100 million years. Calliphora vicina displays a pattern of four rows of bristles on the scutum resembling the postulated ancestral one. We find that sc in Calliphora is expressed in two longitudinal stripes on the medial scutum that prefigure the development of the rows of acrostichal and dorsocentral bristles. This result suggests that a stripe-like expression pattern of sc may be an ancestral feature and may have preceded the evolution of proneural clusters. The implications for the evolution of the cis-regulatory elements responsible for sc expression in the proneural clusters of Drosophila, and function of Pnr are discussed.

Transcriptional heterochrony of scute and changes in bristle pattern between two closely related species of blowfly.

Temporal shifts in the expression of regulatory genes, relative to other events taking place during development, can result in changes in morphology. Such transcriptional heterochrony can introduce dramatic morphological changes that involve rather few genetic events and so has the potential to cause rapid changes during evolution. We have shown previously that stereotyped species-specific bristle patterns on the notum of higher Diptera correlate with changes in the spatial regulation of scute expression. scute encodes a proneural gene required for the development of sensory bristle precursors and is expressed before pupation in discrete domains on the presumptive notum at sites where the macrochaete precursors arise. Thus, for Ceratitis capitata and Calliphora vicina, species separated from Drosophila melanogaster by about 80 and 100 million years respectively, the domains of sc expression differ. In all three species, a second phase of ubiquitous sc expression, after pupation, precedes formation of the microchaete precursors. Here, we describe sc expression in Phormia terranovae, a species belonging to the family Calliphoridae that is closely related to C. vicina. We find that spatial regulation is almost identical between P. terranovae and C. vicina, in spite of their different bristle patterns. The timing of sc expression differs, however, between the two. The first spatially restricted phase of expression is slightly delayed and the second ubiquitous phase remarkably accelerated, such that there is a period of overlap. As a result, the last precursors from the first phase of expression arise at the same time as the first precursors from the second phase of expression and are morphologically indistinguishable from the late-arising microchaetes. These observations illustrate the power of developmental heterochrony in bringing about rapid morphological change.