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.

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.

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.

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.

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.

Neuromuscular Blocking Agent Cisatracurium Attenuates Lung Injury by Inhibition of Nicotinic Acetylcholine Receptor-α1.

BACKGROUND: Neuromuscular blocking agents (NMBAs) bind the nicotinic acetylcholine receptor α1 (nAChRα1) that also contributes to inflammatory signaling. Thus, the author hypothesized that the use of NMBA mitigates lung injury by improving ventilator synchrony and decreasing inflammatory responses. METHODS: Lung injury was induced by intratracheal instillation of hydrogen chloride in rats that were randomized to receive no NMBA with evidence of asynchronous ventilation (noNMBA/aSYNC, n = 10); no NMBA with synchronous ventilation (noNMBA/SYNC, n = 10); cisatracurium (CIS, n = 10); or pancuronium (PAN, n = 10). Mechanical ventilation was set at a tidal volume of 6 ml/kg and positive end-expiratory pressure 8 cm H2O for 3 h. Human lung epithelial, endothelial, and CD14⁺ cells were challenged with mechanical stretch, lipopolysaccharide, lung lavage fluids (bronchoalveolar lavage fluid), or plasma obtained from patients (n = 5) with acute respiratory distress syndrome, in the presence or absence of CIS or small-interfering RNA and small hairpin RNA to attenuate the cell expression of nAChRα1. RESULTS: The use of CIS and PAN improved respiratory compliance (7.2 ± 0.7 in noNMBA/aSYNC, 6.6 ± 0.5 in noNMBA/SYNC, 5.9 ± 0.3 in CIS, and 5.8 ± 0.4 cm H2O/l in PAN; P < 0.05), increased PaO2 (140 ± 54, 209 ± 46, 269 ± 31, and 269 ± 54 mmHg, respectively, P < 0.05), and decreased the plasma levels of tumor necrosis factor-α (509 ± 252 in noNMBA, 200 ± 74 in CIS, and 175 ± 84 pg/ml in PAN; P < 0.05) and interleukin-6 (5789 ± 79, 1608 ± 534, and 2290 ± 315 pg/ml, respectively; P < 0.05). The use of CIS and PAN or silencing the receptor nAChRα1 resulted in decreased cytokine release in the human cells in response to a variety of stimuli mentioned earlier. CONCLUSIONS: The use of NMBA is lung protective through its antiinflammatory properties by blocking the nAChRα1.

Regional mapping of myocardial hibernation phenotype in idiopathic end-stage dilated cardiomyopathy.

Myocardial hibernation (MH) is a well-known feature of human ischaemic cardiomyopathy (ICM), whereas its presence in human idiopathic dilated cardiomyopathy (DCM) is still controversial. We investigated the histological and molecular features of MH in left ventricle (LV) regions of failing DCM or ICM hearts. We examined failing hearts from DCM (n = 11; 41.9 ± 5.45 years; left ventricle-ejection fraction (LV-EF), 18 ± 3.16%) and ICM patients (n = 12; 58.08 ± 1.7 years; LVEF, 21.5 ± 6.08%) undergoing cardiac transplantation, and normal donor hearts (N, n = 8). LV inter-ventricular septum (IVS) and antero-lateral free wall (FW) were transmurally (i.e. sub-epicardial, mesocardial and sub-endocardial layers) analysed. LV glycogen content was shown to be increased in both DCM and ICM as compared with N hearts (P < 0.001), with a U-shaped transmural distribution (lower values in mesocardium). Capillary density was homogenously reduced in both DCM and ICM as compared with N (P < 0.05 versus N), with a lower decrease independent of the extent of fibrosis in sub-endocardial and sub-epicardial layers of DCM as compared with ICM. HIF1-α and nestin, recognized ischaemic molecular hallmarks, were similarly expressed in DCM-LV and ICM-LV myocardium. The proteomic profile was overlapping by ~50% in DCM and ICM groups. Morphological and molecular features of MH were detected in end-stage ICM as well as in end-stage DCM LV, despite epicardial coronary artery patency and lower fibrosis in DCM hearts. Unravelling the presence of MH in the absence of coronary stenosis may be helpful to design a novel approach in the clinical management of DCM.

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.

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.

Detection of human cytomegalovirus in transbronchial biopsies from lung transplant recipients.

The role of human cytomegalovirus (HCMV) in lung transplantation (LT) and drawbacks related to viral quantification in bronchoalveolar lavage (BAL) underline the potential usefulness of investigating other specimens. Thirty-three LT recipients were prospectively studied by HCMV quantitative real time PCR on matched transbronchial biopsy (TBB), BAL, and whole blood specimens. Overall, 27/33 patients turned out HCMV-positive in at least one specimen: 7.1 %, 37.1 %, and 13.5 % of TBB, BAL, and blood samples, respectively. No significant association between HCMV on all types of specimens and acute rejection, lymphocytic bronchiolitis, bronchiolitis obliterans and bronchiolitis obliterans syndrome was found. HCMV pneumonia was associated to HCMV detection on TBB (p = 0.003) and whole blood (p = 0.008), not on BAL (p = 0.47). The highest mean viral load was detected in TBB from cases with HCMV pneumonia in comparison to all other cases, suggesting the potential use of HCMV investigation in TBB for evaluating posttransplant complications.

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.

DNA methyltransferase 3b (DNMT3b), tumor tissue DNA methylation, Gleason score, and prostate cancer mortality: investigating causal relationships.

PURPOSE: Aberrant DNA methylation plays a role in prostate cancer progression. We studied the relationships among DNA methyltransferase (DNMT) genotype, DNA methylation, Gleason score, and mortality in two cohorts of prostate cancer patients, previously reported with associations between DNA methylation in GSTP1, APC, and RUNX3 and prostate cancer mortality. Herein, we considered possible causal relationships between the studied variables, assuming that (1) DNMT activity affects tumor tissue methylation, (2) methylation status affects tumor morphology, and thus the Gleason score, and (3) DNA methylation affects mortality via Gleason score. METHODS: The cohorts comprised 438 patients diagnosed at one Italian pathology ward before 1997, with DNA obtained from paraffin-embedded tumor tissues. The polymorphism rs406193 in the DNMT3b gene was assessed by allele discrimination in real-time PCR. According to the assumed causal model, we analyzed the effects of rs406193 (T carriers vs others) on the Gleason score without adjusting for gene methylation, and the effects of rs406193 on gene methylation and prostate cancer mortality without adjusting for Gleason score. RESULTS: We found no evidence of association between T carriers and the number of methylated genes. However, T carriers had reduced risk of a Gleason score 8+ (odds ratio = 0.57, 95 % CI 0.39-0.85), and a hazard ratio of 0.81 (0.61-1.09) of dying from prostate cancer, which would have been erroneously estimated of 0.93 if adjusted for Gleason score. CONCLUSIONS: These findings provide clues on the role of a DNMT3b SNP in prostate cancer progression and illustrate the importance of considering possible causal relationships in the analyses.

Phosphoinositide-3 kinase gamma activity contributes to sepsis and organ damage by altering neutrophil recruitment.

RATIONALE: Sepsis is a leading cause of death in the intensive care unit, characterized by a systemic inflammatory response (SIRS) and bacterial infection, which can often induce multiorgan damage and failure. Leukocyte recruitment, required to limit bacterial spread, depends on phosphoinositide-3 kinase γ (PI3Kγ) signaling in vitro; however, the role of this enzyme in polymicrobial sepsis has remained unclear. OBJECTIVES: This study aimed to determine the specific role of the kinase activity of PI3Kγ in the pathogenesis of sepsis and multiorgan damage. METHODS: PI3Kγ wild-type, knockout, and kinase-dead mice were exposed to cecal ligation and perforation-induced sepsis and assessed for survival; pulmonary, hepatic, and cardiovascular damage; coagulation derangements; systemic inflammation; bacterial spread; and neutrophil recruitment. Additionally, wild-type mice were treated either before or after the onset of sepsis with a PI3Kγ inhibitor and assessed for survival, neutrophil recruitment, and bacterial spread. MEASUREMENTS AND MAIN RESULTS: Both genetic and pharmaceutical PI3Kγ kinase inhibition significantly improved survival, reduced multiorgan damage, and limited bacterial decompartmentalization, while modestly affecting SIRS. Protection resulted from both neutrophil-independent mechanisms, involving improved cardiovascular function, and neutrophil-dependent mechanisms, through reduced susceptibility to neutrophil migration failure during severe sepsis by maintaining neutrophil surface expression of the chemokine receptor, CXCR2. Furthermore, PI3Kγ pharmacological inhibition significantly decreased mortality and improved neutrophil migration and bacterial control, even when administered during established septic shock. CONCLUSIONS: This study establishes PI3Kγ as a key molecule in the pathogenesis of septic infection and the transition from SIRS to organ damage and identifies it as a novel possible therapeutic target.

Effect of CMV-immunoglobulins (cytotect biotest) prophylaxis on CMV pneumonia after lung transplantation.

Lung transplant (LT) recipients among solid organ transplant recipients are at high risk for cytomegalovirus (CMV) infections. We evaluated the effect of CMV-Immunoglobulins (CMV-IG) (Cytotect Biotest) on CMV pneumonia diagnosed in 303 follow-up transbronchial biopsies (TBB) of lung transplant recipients. 24 patients (control group, 155 TBB from 1999 to 2002) received acyclovir for 24 months and 33 recipients (study group, 148 TBB from 2003 to 2008) received a combined CMV prophylaxis consisting of CMV-IG (Cytotect Biotest) for 12 months and a short Ganciclovir or Valganciclovir therapy from 21th to 42th postoperative day followed by acyclovir up to 24 months. In our study the percentage of pneumonia at first month TBB was similar in the study group vs the control group, 9.1% (3/33) vs 8.3% (2/24), p=0.9 ns, but after the first month the percentage was significantly lower in the study group in the first year at follow-up TBB, 1% (1/99) vs 6.4% (5/78), p=0.048, and in first two years follow-up TBB, 0.8% (1/122) vs 6.5% 8/124), p=0.018 (STATISTICAL ANALYSIS: Chi-square test for proportion differences). Our data suggest a strong efficacy of CMV-IG prophylaxis in reducing CMV pneumonia after first month in lung transplant recipients.

Quantitative detection of HHV-6 and HHV-7 in transbronchial biopsies from lung transplant recipients.

The occurrence and significance of HHV-6 and HHV-7 were investigated in pulmonary tissue from lung transplant recipients. Eighty-seven transbronchial biopsies from 30 patients were studied by quantitative real-time PCR; the association with histopathological features was investigated. HHV-6 and HHV-7-DNA were detected in 6.9% and 9.2% transbronchial biopsies, respectively. A significant association between HHV-6 detection on transbronchial biopsies and interstitial pneumonia was found, in contrast to the lack of association between viral detection on bronchoalveolar lavage and any histopathological feature. No association was evidenced in terms of acute and chronic rejection. The finding of HHV-6 and/or HHV-7-DNA positivity in all the cases with ischemia-reperfusion injury suggests a possible role in favouring ?-herpesviruses reactivation, as previously described for HCMV in renal transplantation.

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.

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.

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.