Dickkopf-1 (DKK1) drives growth and metastases in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is associated with a poor prognosis and remains an incurable fatal disease. Therefore, the identification of molecular markers involved in cancer progression is urgently needed to develop more-effective therapies. The present study investigated the role of the Wnt signaling modulator Dickkopf-1 (DKK1) in the growth and metastatic progression of mCRPC. DKK1 silencing through siRNA and deletion via CRISPR/Cas9 editing were performed in two different metastatic castration-resistant prostate cancer cell lines (PC3 and DU145). A xenograft tumor model was used to assess tumor growth and metastases. In in vitro experiments, both DKK1 silencing and deletion reduced cell growth and migration of both cell lines. DKK1 knockout clones (DKK1-KO) exhibited cell cycle arrest, tubulin reorganization, and modulation of tumor metastasis-associated genes. Furthermore, in DKK1-KO cells, E-cadherin re-expression and its membrane co-localization with ß-catenin were observed, contributing to reduced migration; Cadherin-11, known to increase during epithelial-mesenchymal transition, was down-regulated in DKK1-KO cells. In the xenograft mouse model, DKK1 deletion not only reduced tumor growth but also inhibited the formation of lung metastases. In conclusion, our findings support the key role of DKK1 in the growth and metastatic dissemination of mCRPC, both in vitro and in vivo.

Correlation between high-resolution computed tomography appearance and histopathological features in the diagnosis of interstitial lung diseases. A real-life study.

BACKGROUND: According to current guidelines, a surgical biopsy is rarely required when a high-confidence radiologic interstitial lung disease (ILD) diagnosis is made on thin-section high-resolution computed tomography (HRCT). Nevertheless, disowning HRCT scans diagnosed by biopsy are more common than presumed. Our study aimed to describe the concordance rate between HRCT scans and pathological diagnoses of ILDs obtained by surgical biopsy. The current guideline suggests the use of surgical lung biopsy (SLB) in patients with newly detected ILD of unknown cause. METHODS: Patients who underwent mini-invasive surgical biopsies for interstitial lung diseases from January 2018 to August 2022 were analyzed. The HRCT scans were reviewed by an observer blinded to the patient's clinical information. The concordance between histological and HRCT-scan were assessed. RESULTS: Data from 104 patients with uncertain low confidence diagnosis of interstitial lung diseases at HRCT were analyzed. Most of the patients are male (65; 62.5%). The more frequent HRCT pattern were: alternative diagnoses (46; 44.23%), UIP probable (42; 40.38%), UIP indeterminate (7; 6.73%), and non-specific interstitial pneumonia (NSIP) (9, 8.65%). The more common histological diagnosis was UIP definite (30; 28.84%), hypersensitivity pneumonia [HP](19; 18.44%), NSIP (15; 14.42%), sarcoidosis (10; 9.60%). In 7 (20%) cases, the final pathological finding denies HRCT-scans diagnoses; indeed, a moderate agreement was observed between HRCT-scan findings and the definitive histological diagnosis (kappa index: 0.428). CONCLUSIONS: HRCT-scan has limitations if the objective is to define interstitial lung diseases accurately. Consequently, pathological assessment should be taken into account in order to provide more accurate tailored treatment strategies because the risk is to wait from 12 to 24 months to ascertain if the ILD will be treatable as progressive pulmonary fibrosis (PPF). Undeniably true, video-assisted surgical lung biopsy (VASLB) with endotracheal intubation and mechanical ventilation is associated with a risk of mortality and morbidity that is far from nil. Nevertheless, in recent years a VASLB approach performed in awake subjects under loco-regional anesthesia (awake-VASLB) has been suggested as an effective method to obtain a highly confident diagnosis in patients with diffuse pathologies of the lung parenchyma.

Robot-assisted PSMA-radioguided Surgery to Assess Surgical Margins and Nodal Metastases in Prostate Cancer Patients: Report on Three Cases Using an Intraoperative PET-CT Specimen Imager.

INTRODUCTION: The aim of this feasibility study was to test the intraoperative use of this brand-new specimen PET/CT to guide robot-assisted radical prostatectomy and pelvic lymph node dissection. MATERIALS AND METHODS: Three cases of robot-assisted radical prostatectomy and pelvic lymph node dissection were performed with intraoperative use of the specimen imager. Surgeries were performed with Da Vinci Xi robot. An intravenous injection of 68Ga-PSMA-11 was performed in the OR and after complete excision, the specimens were analyzed with the imager. RESULTS: The average nodal yield was 17.3 (5.8 SD) nodes per patient. Specimen PET/CT images showed a focal uptake in a metastatic node (TBR 13.6), and no uptake or diffuse, faint uptake in negative nodes (TBR range: 1-5.3). The specimen imager provided intraoperative PET/CT images that clearly showed negative surgical margins in two patients, whereas the results were uncertain in a locally advanced case. CONCLUSION: The intraoperative use of the specimen PET/CT imager is safe and feasible and could improve the evaluation of prostate surgical margins and lymph node status.

Is there life on the airway tree? A pilot study of bronchial cell vitality and tissue morphology in the ex vivo lung perfusion (EVLP) era of lung transplantation.

BACKGROUND: Ex vivo lung perfusion (EVLP) is a relevant procedure to increase the lung donor pool but could potentially increase the airway tree ischemic injury risk. METHODS: This study aimed to evaluate the direct effect of EVLP on the airway tree by evaluating bronchial cell vitality and tissue signs of injury on a series of 117 bronchial rings collected from 40 conventional and 19 EVLP-treated lung grafts. Bronchial rings and related scraped bronchial epithelial cells were collected before the EVLP procedure and surgical anastomosis. RESULTS: The preimplantation interval was significantly increased in the EVLP graft group (p < 0.01). Conventional grafts presented cell viability percentages of 47.07 ± 23.41 and 49.65 ± 21.25 in the first and second grafts which did not differ significantly from the EVLP group (first graft 50.54 ± 25.83 and second graft 50.22 ± 20.90 cell viability percentage). No significant differences in terms of histopathological features (edema, inflammatory infiltrate, and mucosa ulceration) were observed comparing conventional and EVLP samples. A comparison of bronchial cell viability and histopathology of EVLP samples retrieved at different time intervals revealed no significant differences. Accordingly, major bronchial complications after lung transplant were not observed in both groups. CONCLUSIONS: Based on these data, we observed that EVLP did not significantly impact bronchial cell vitality and airway tissue preservation nor interfere with bronchial anastomosis healing, further supporting it as a safe and useful procedure.

Validation of a Simple, Rapid, and Cost-Effective Method for Acute Rejection Monitoring in Lung Transplant Recipients.

Despite advances in immunosuppression therapy, acute rejection remains the leading cause of graft dysfunction in lung transplant recipients. Donor-derived cell-free DNA is increasingly being considered as a valuable biomarker of acute rejection in several solid organ transplants. We present a technically improved molecular method based on digital PCR that targets the mismatch between the recipient and donor at the HLA-DRB1 locus. Blood samples collected sequentially post-transplantation from a cohort of lung recipients were used to obtain proof-of-principle for the validity of the assay, correlating results with transbronchial biopsies and lung capacity tests. The results revealed an increase in dd-cfDNA during the first 2 weeks after transplantation related to ischemia-reperfusion injury (6.36 ± 5.36%, p < 0.0001). In the absence of complications, donor DNA levels stabilized, while increasing again during acute rejection episodes (7.81 ± 12.7%, p < 0.0001). Respiratory tract infections were also involved in the release of dd-cfDNA (9.14 ± 15.59%, p = 0.0004), with a positive correlation with C-reactive protein levels. Overall, the dd-cfDNA percentages were inversely correlated with the lung function values measured by spirometry. These results confirm the value of dd-cfDNA determination during post-transplant follow-up to monitor acute rejection in lung recipients, achieved using a rapid and inexpensive approach based on the HLA mismatch between donor and recipient.

HLA-DRB1 mismatch-based identification of donor-derived cell free DNA (dd-cfDNA) as a marker of rejection in heart transplant recipients: A single-institution pilot study.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. METHODS: In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. RESULTS: Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. CONCLUSIONS: These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.

Natural history of widespread high grade prostatic intraepithelial neoplasia and atypical small acinar proliferation: should we rebiopsy them all?

OBJECTIVE: To evaluate the premalignant potential of high-grade prostatic intraepithelial neoplasia (HGPIN) and atypical small acinar proliferation (ASAP). METHODS: Patients diagnosed with monofocal HGPIN (mHGPIN), widespread HGPIN (≥4 cores, wHGPIN) and/or ASAP who underwent at least one rebiopsy during their follow-up, were enrolled. All enrollment biopsies underwent central pathologic revision. Risks for PCa were estimated using Fine and Gray method for competing risk. RESULTS: Pathologic revision changed the original diagnosis in 32.3% of cases. Among 336 cases enrolled, PCa was diagnosed in 164 (48.8%), and more specifically in 20 (30.3%) mHGPIN, 10 (34.5%) wHGPIN, 101 (54.0%) ASAP, and 33 (61.1%) HGPIN + ASAP (mean follow-up 124 months). Most PCa were Gleason score 6(3 + 3) (51.0%) and 7(3 + 4) (34.3%). On multivariate analysis, HGPIN + ASAP (HR 2.76, p < 0.001) and ASAP alone (HR 2.41, p < 0.001) were the only lesions significantly associated with PCa development. Of all cancers detected, 64.3% were at first rebiopsy. A rebiopsy performed within 3 months after ASAP diagnosis had a 45% chance of finding PCa. At Kaplan-Meier survival curves, median PCa-free survival was 48.1 months for HGPIN + ASAP and 64.9 months for ASAP (p 0.0005 at Log-rank test). At 1 year, 70% of HGPIN + ASAP, 73% of ASAP, 89% of wHGPIN, and 84% of mHGPIN were PCa-free. CONCLUSION: The diagnosis of ASAP and HGPIN strongly relies on the expertise of dedicated uro-pathologists. Finding of ASAP is a strong risk factor for a subsequent PCa diagnosis, advising a rebiopsy, possibly within 3 months. m/wHGPIN should not be routinely rebiopsied.

Gross Specimen Handling Procedures Do Not Impact the Occurrence of Spread Through Air Spaces (STAS) in Lung Cancer.

Spread Through Air Spaces (STAS) is a form of invasion characterized by neoplastic cell dissemination in the lung parenchyma surrounding the outer edge of the tumor. Its possible artifactual origin is widely debated in the literature. The aim of this study is to investigate the potential impact of gross sampling procedures in causing STAS. A prospective series of 51 surgical lung specimens was collected (35 adenocarcinomas, 68.6%; 13 squamous cell carcinomas, 25.5%; 2 large-cell neuroendocrine carcinomas, 3.9%; 1 atypical carcinoid, 2%). The fresh tissue was sectioned with a new and clean blade for each cut, to obtain a tissue slice comprising the upper lung parenchyma, the tumor, and the lower parenchyma. This slice was cut in half and separately processed. The same procedure was repeated in the residual (specular) specimen after formalin fixation. STAS was identified in 33/51 (64.7%) cases, the predominant pattern being cluster formation (29 cases, 87.9%), the remaining 4 cases having single-cell invasion. Comparing STAS detection in upper and lower lung parenchyma areas (ie, before and after the blade crossed the tumor), no significant preferential STAS distribution was observed, indeed being almost overlapping (60.6% and 63.6% for fresh and 61.3% and 65.6% for fixed tissues, respectively). There was no difference between STAS occurrence in freshly cut and fixed corresponding samples. These findings indicate that STAS is not a pathologist-related artifactual event because of knife transportation of tumor cells during gross specimen handling and support the notion that it is a phenomenon preexisting to surgical tissue processing.

Kinomic profiling of tumour xenografts derived from patients with non-small cell lung cancer confirms their fidelity and reveals potentially actionable pathways.

INTRODUCTION: High fidelity between non-small cell lung cancer (NSCLC) primary tumours and patient-derived tumour xenografts (PDTXs) is of paramount relevance to spur their application. Extensive proteomic and kinomic analysis of these preclinical models are missing and may inform about their functional status, in terms of phosphopeptides and hyperactive signalling pathways. METHODS: We investigated tumour xenografts derived from patients with NSCLC to identify hyperactive signalling pathways. Fresh tumour fragments from 81 NSCLC surgical samples were implanted in Nod/Scid/Gamma mice, and engrafted tumours were compared with primary specimens by morphology, immunohistochemistry, gene mutation analyses, and kinase activity profiling. Four different tyrosine and serine/threonine kinase inhibitors were tested against primary tumour and PDTX lysates using the PamGene peptide microarray platform. RESULTS: The engraftment rate was 33%, with successful engraftment being more associated with poor clinical outcomes. Genomic profiles led to the recognition of hotspot mutations, some of which were initially undetected in donor samples. Kinomic analyses showed that characteristics of primary tumours were retained in PDTXs, and tyrosine kinase inhibitors (TKIs) responses of individual PDTX lines were either expected, based on the genetic status, or alternatively defined suitable targets unpredictable by single-genome fingerprints. CONCLUSIONS: Collectively, PDTXs mostly resembled their parental NSCLC. Combining genomic and kinomic analyses of tumour xenografts derived from patients with NSCLC, we identified patients' specific targetable pathways, confirming PDTXs as a preclinical tool for biomarker identification and therapeutic algorithm'' improvement.

Modulating tumor reactive stroma by extracorporeal shock waves to control prostate cancer progression.

BACKGROUND: Prostate cancer is the second most common cancer worldwide. Tumor microenvironment is composed of activated fibroblasts, the so called carcinoma-associated fibroblasts (CAFs). They express high levels of α-smooth muscle actin (α-SMA) and type I collagen (COL1), and support proliferation and migration of tumor epithelial cells. Extracorporeal shock waves (ESWs), acoustic waves, are effective in the treatment of hypertrophic scars, due to their ability to modulate fibrosis. Based on this rationale, the study evaluated the effects of ESWs on CAF activation and the influence of ESW-treated CAFs on the growth and migration of epithelial prostatic carcinoma cells. METHODS: Primary cultures of CAFs (n = 10) were prepared from tumors of patients undergoing surgery for high-risk prostate carcinoma. CAFs were treated with ESWs (energy levels: 0.32 mJ/mm2 , 1000 pulses; 0.59 mJ/mm2 , 250 pulses). After treatment, the messenger RNA and protein levels of the stromal activation markers α-SMA and COL1 were determined. Subsequently, two different stabilized cell lines (PC3 and DU145) of androgen-resistant prostate cancer were treated with the conditioned media produced by ESW-treated CAFs. At different times, viability and migration of PC3 and DU145 cells were evaluated. Viability was also assessed by coculture system using CAFs and PC3 or DU145 cells. RESULTS: ESWs reduced gene expression and protein level of α-SMA and COL1 in CAFs. The treatment of PC3 and DU145 with conditioned media of ESW-treated CAFs determined a reduction of their growth and invasive potential. Coculture systems between ESW-treated CAFs and PC3 or DU145 cells confirmed the epithelial cell number reduction. CONCLUSIONS: This in vitro study demonstrates for the first time that ESWs are able to modulate the activation of prostate CAFs in favor of a less "reactive" stroma, with consequent slowing of the growth and migration of prostate cancer epithelial cells. However, only further studies to be performed in vivo will confirm the possibility of using this new therapy in patients with prostate cancer.

Role of Immunocytochemistry in the Cytological Diagnosis of Pulmonary Tumors.

Pulmonary cytology is a challenging diagnostic tool, and it is usually evaluated considering medical history and radiological findings in order to reach an accurate diagnosis. Since the majority of lung cancer patients have an advanced stage at diagnosis, a cytological specimen is frequently the only material available for diagnosis and further prognostic/predictive marker determination. Several types of specimens can be obtained from the respiratory system (including sputum, bronchoalveolar lavage, bronchial brushing, fine needle aspiration, and pleural fluid) with different technical preclinical management protocols and different diagnostic yields. Immunocytochemistry (ICC) has a pivotal role in the determination of diagnostic, prognostic, and predictive markers. Therefore, limited cytology samples are to be used with a cell-sparing approach, to allow both diagnostic ICC evaluation as well as predictive marker assessment by ICC or specific molecular assays. In this review, we describe the most common ICC markers used for the diagnosis and prognostic/predictive characterization of thoracic tumors in different cytological specimens.

DNA methylation in repeat negative prostate biopsies as a marker of missed prostate cancer.

BACKGROUND: Men often undergo repeat prostate biopsies because of suspicion of missed cancer. We assessed if (i) methylation of selected genes in prostate tissue vary with aging and (ii) methylation alterations in repeat biopsies predict missed prostate cancer. METHODS: We conducted a case-control study among men who underwent at least two negative prostate biopsies followed by a sampling either positive (cases n = 111) or negative (controls n = 129) for prostate cancer between 1995 and 2014 at the University Hospital (Turin, Italy). Two pathology wards were included for replication purposes. We analyzed methylation of GSTP1, APC, PITX2, C1orf114, GABRE, and LINE-1 in the first two negative biopsies. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of the association between genes methylation and prostate cancer. RESULTS: Age at biopsy and time interval between the two negative biopsies were not associated with methylation levels of the selected genes in neither cases nor controls. GSTP1 methylation in the first and in the second negative biopsy was associated with prostate cancer detection [OR per 1% increase: 1.14 (95% CI 1.01-1.29) for the second biopsy and 1.21 (95% CI 1.07-1.37) for the highest methylation level (first or second biopsy)]. A threshold > 10% for GSTP1 methylation corresponded to a specificity of 0.98 (positive likelihood ratio 7.87). No clear association was found for the other genes. Results were consistent between wards. CONCLUSIONS: Our results suggest that GSTP1 methylation in negative prostate biopsies is stable over time and can predict missed cancer with high specificity.

Feasibility of lung microdialysis to assess metabolism during clinical ex vivo lung perfusion.

BACKGROUND: Lung metabolism during ex vivo lung perfusion (EVLP) is increasingly studied. Microdialysis (MD) allows metabolic monitoring by sampling parenchymal interstitial fluid. This study investigated lung metabolism using MD during EVLP and evaluated whether microdialysate metabolites could improve selection and discriminate outcome of donor lungs. METHODS: MD monitoring was used during 14 clinical EVLP procedures. Paired microdialysate and perfusate samples were analyzed for glucose, lactate, pyruvate, glutamate, and the lactate/pyruvate (L/P) ratio, and values that best discriminated an unfavorable outcome were determined. Outcome was defined as unfavorable (lungs not transplanted or transplanted with primary graft dysfunction at 72 hours ≥ 2) or favorable (lungs transplanted with primary graft dysfunction < 2). RESULTS: Microdialysate markers and the perfusate L/P ratio could discriminate unfavorable outcome with sensitivity and specificity of 0.85 and 0.81 for MD glutamate > 18.4 µmol/liter, 0.81 and 0.74 for MD lactate > 685 µmol/liter, 0.92 and 0.75 for MD glucose > 530 µmol/liter, 0.85 and 0.65 for MD pyruvate > 25 µmol/liter, and 0.73 and 0.67 for perfusate L/P ratio > 24.17. All microdialysate markers, perfusate and microdialysate L/P ratio, and perfusate lactate discriminated outcome when we limited analysis only to transplanted lungs. CONCLUSIONS: We report the use of MD to evaluate lung metabolism during clinical EVLP, demonstrating that MD metabolites can contribute to selection of reconditioned lungs and discriminate early outcome after transplantation. Furthermore, glutamate as a marker of lung injury during EVLP is proposed and could hence be used as a potential target for future therapies.

Single-nucleotide polymorphisms in DNMT3B gene and DNMT3B mRNA expression in association with prostate cancer mortality.

BACKGROUND: Germline variants in DNA methyltransferase 3B (DNMT3B) may influence DNMT3B enzymatic activity, which, in turn, may affect cancer aggressiveness by altering DNA methylation. METHODS: The study involves two Italian cohorts (NTAT cohort, n = 157, and 1980s biopsy cohort, n = 182) and two U.S. cohorts (Health Professionals Follow-Up Study, n = 214, and Physicians' Health Study, n = 298) of prostate cancer (PCa) patients, and a case-control study of lethal (n = 113) vs indolent (n = 290) PCa with DNMT3B mRNA expression data nested in the U.S. cohorts. We evaluated the association between: three selected DNMT3B variants and global DNA methylation using linear regression in the NTAT cohort, the three DNMT3B variants and PCa mortality using Cox proportional hazards regression in all cohorts, and DNMT3B expression and lethal PCa using logistic regression, with replication in publicly available databases (TCGA, n = 492 and MSKCC, n = 140). RESULTS: The TT genotype of rs1569686 was associated with LINE-1 hypomethylation in tumor tissue (ß = -2.71, 95% CI: -5.41, -0.05). There was no evidence of association between DNMT3B variants and PCa mortality. DNMT3B expression was consistently associated with lethal PCa in the two U.S. cohorts (3rd vs 1st tertile, combined cohorts: OR = 2.04, 95% CI: 1.13, 3.76); the association was replicated in TCGA and MSKCC data (3rd vs 1st tertile, TCGA: HR = 3.00, 95% CI: 1.78, 5.06; MSKCC: HR = 2.22, 95% CI: 1.01, 4.86). CONCLUSIONS: Although there was no consistent evidence of an association between DNMT3B variants and PCa mortality, the TT genotype of rs1569686 was associated with LINE-1 hypomethylation in tumor tissue and DNMT3B mRNA expression was associated with an increased risk of lethal PCa.

Identification and Modulation of Microenvironment Is Crucial for Effective Mesenchymal Stromal Cell Therapy in Acute Lung Injury.

Rationale: There are controversial reports on applications of mesenchymal stromal cells (MSCs) in patients with acute respiratory distress syndrome (ARDS). Objectives: We hypothesized that lung microenvironment was the main determinant of beneficial versus detrimental effects of MSCs during ARDS. Methods: Lung proteome was profiled in three models of injury induced by acid instillation and/or mechanical ventilation in mice. Human gene of glutathione peroxidase-1 was delivered before MSC administration; or MSCs carrying human gene of IL-10 or hepatocyte growth factor were administered after lung injury. An inhibitory cocktail against IL-6, fibronectin, and oxidative stress was used in in vitro studies using human small airway epithelial cells and human MSCs after exposure to plasma of patients with ARDS. Measurements and Main Results: Distinct proteomic profiles were observed in three lung injury models. Administration of MSCs protected lung from ventilator-induced injury, whereas it worsened acid-primed lung injuries associated with fibrotic development in lung environment that had high levels of IL-6 and fibronectin along with low antioxidant capacity. Correction of microenvironment with glutathione peroxidase-1, or treatment with MSCs carrying human gene of IL-10 or hepatocyte growth factor after acid-primed injury, reversed the detrimental effects of native MSCs. Proteomic profiles obtained in the mouse models were also similarly observed in human ARDS. Treatment with the inhibitory cocktail in samples of patients with ARDS retained protective effects of MSCs in small airway epithelial cells. Conclusions: MSCs can be beneficial or detrimental depending on microenvironment at the time of administration. Identification of potential beneficiaries seems to be crucial to guide MSC therapy in ARDS.

Cell of origin markers identify different prognostic subgroups of lung adenocarcinoma.

Strong prognostic markers able to stratify lung adenocarcinoma (ADC) patients are lacking. We evaluated whether a six-immunohistochemical markers panel (TTF1, SP-A, Napsin A, MUC5AC, CDX2 and CK5), defining the putative neoplastic "cell of origin," allows to identify prognostic subgroups among lung ADC. We screened a large cohort of ADC specimens (2003-2013) from Torino Institutional Repository identifying: (i) marker positivity by immunohistochemistry, (ii) main morphological appearance by light microscopy, (iii) presence of "hotspot" mutations of candidate genes by Sequenom technology. To evaluate possible predictors of survival and time to recurrence, uni- and multivariable-adjusted comparisons were performed. We identified 4 different subgroups: "alveolar," "bronchiolar," "mixed" and "null type." Alveolar-differentiated ADC were more common in young (P=.065), female (P=.083) patients, frequently harboring EGFR-mutated (P=.003) tumors with acinar pattern (P<.001). Bronchiolar-differentiated ADC were more associated with mucinous and solid pattern (P<.001), higher degree of vascular invasion (P=.01) and KRAS gene mutations (P=.07). Bronchiolar, mixed, and null types were independent negative predictors for overall survival, and the latter two had a shorter time to recurrence. This "Cell of Origin" classifier is more predictable than morphology and genetics and is an independent predictor of survival on a multivariate analysis.

LINE-1 methylation status in prostate cancer and non-neoplastic tissue adjacent to tumor in association with mortality.

Aberrant DNA methylation seems to be associated with prostate cancer behavior. We investigated LINE-1 methylation in prostate cancer and non-neoplastic tissue adjacent to tumor (NTAT) in association with mortality from prostate cancer. We selected 157 prostate cancer patients with available NTAT from 2 cohorts of patients diagnosed between 1982-1988 and 1993-1996, followed up until 2010. An association between LINE-1 hypomethylation and prostate cancer mortality in tumor was suggested [hazard ratio per 5% decrease in LINE-1 methylation levels: 1.40, 95% confidence interval (CI): 0.95-2.01]. After stratification of the patients for Gleason score, the association was present only for those with a Gleason score of at least 8. Among these, low (<75%) vs. high (>80%) LINE-1 methylation was associated with a hazard ratio of 4.68 (95% CI: 1.03-21.34). LINE-1 methylation in the NTAT was not associated with prostate cancer mortality. Our results are consistent with the hypothesis that tumor tissue global hypomethylation may be a late event in prostate cancerogenesis and is associated with tumor progression.

Intratracheal Administration of Small Interfering RNA Targeting Fas Reduces Lung Ischemia-Reperfusion Injury.

OBJECTIVES: Lung ischemia-reperfusion injury is the main cause of primary graft dysfunction after lung transplantation and results in increased morbidity and mortality. Fas-mediated apoptosis is one of the pathologic mechanisms involved in the development of ischemia-reperfusion injury. We hypothesized that the inhibition of Fas gene expression in lungs by intratracheal administration of small interfering RNA could reduce lung ischemia-reperfusion injury in an ex vivo model reproducing the procedural sequence of lung transplantation. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University research laboratory. SUBJECTS: C57/BL6 mice weighing 28-30 g. INTERVENTIONS: Ischemia-reperfusion injury was induced in lungs isolated from mice, 48 hours after treatment with intratracheal small interfering RNA targeting Fas, control small interfering RNA, or vehicle. Isolated lungs were exposed to 6 hours of cold ischemia (4°C), followed by 2 hours of warm (37°C) reperfusion with a solution containing 10% of fresh whole blood and mechanical ventilation with constant low driving pressure. MEASUREMENTS AND MAIN RESULTS: Fas gene expression was significantly silenced at the level of messenger RNA and protein after ischemia-reperfusion in lungs treated with small interfering RNA targeting Fas compared with lungs treated with control small interfering RNA or vehicle. Silencing of Fas gene expression resulted in reduced edema formation (bronchoalveolar lavage protein concentration and lung histology) and improvement in lung compliance. These effects were associated with a significant reduction of pulmonary cell apoptosis of lungs treated with small interfering RNA targeting Fas, which did not affect cytokine release and neutrophil infiltration. CONCLUSIONS: Fas expression silencing in the lung by small interfering RNA is effective against ischemia-reperfusion injury. This approach represents a potential innovative strategy of organ preservation before lung transplantation.

The Influence of Tissue Ischemia Time on RNA Integrity and Patient-Derived Xenografts (PDX) Engraftment Rate in a Non-Small Cell Lung Cancer (NSCLC) Biobank.

INTRODUCTION: Bio-repositories are invaluable resources to implement translational cancer research and clinical programs. They represent one of the most powerful tools for biomolecular studies of clinically annotated cohorts, but high quality samples are required to generate reliable molecular readouts and functional studies. The objective of our study was to define the impact of cancer tissue ischemia time on RNA and DNA quality, and for the generation of Patient-Derived Xenografts (PDXs). METHODS: One-hundred thirty-five lung cancer specimens were selected among our Institutional BioBank samples. Associations between different warm (surgical) and cold (ex-vivo) ischemia time ranges and RNA quality or PDXs engraftment rates were assessed. RNA quality was determined by RNA integrity number (RINs) values. Fresh viable tissue fragments were implanted subcutaneously in NSG mice and serially transplanted. RESULTS: RNAs with a RIN>7 were detected in 51% of the sample (70/135), with values of RIN significantly lower (OR 0.08, P = 0.01) in samples preserved for more than 3 hours before cryopreservation. Higher quality DNA samples had a concomitant high RIN. Sixty-three primary tumors (41 adenocarcinoma) were implanted with an overall engraftment rate of 33%. Both prolonged warm (>2 hours) and ex-vivo ischemia time (>10 hours) were associated to a lower engraftment rate (OR 0.09 P = 0.01 and OR 0.04 P = 0.008, respectively). CONCLUSION: RNA quality and PDXs engraftment rate were adversely affected by prolonged ischemia times. Proper tissue collection and processing reduce failure rate. Overall, NSCLC BioBanking represents an innovative modality, which can be successfully executed in routine clinical settings, when stringent Standard Operating Procedures are adopted.