Luigi Santambrogio

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells

Summary Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Does Surgery Improve Survival of Patients with Malignant Pleural Mesothelioma?: A Multicenter Retrospective Analysis of 1365 Consecutive Patients

Background: Surgery with pleurectomy/decortication (P/D) or extrapleural pneumonectomy (EPP) can be an option for selected patients with resectable malignant pleural mesothelioma (MPM). The aim of this study was to investigate the impact of surgical treatment on the outcome of patients with MPM. Methods: We retrospectively reviewed data from 1365 consecutive patients with histologically proven MPM, treated from 1982 to 2012 in six Institutions. Patients received chemotherapy alone (n = 172), best supportive care (n = 690), or surgical treatment (n = 503), by either P/D (n = 202) or EPP (n = 301) with or without chemotherapy. Results: After a median follow-up of 6.7 years (range, 1.1–14.8), 230 patients (16.8%) were alive; median survival for patients who received palliative treatment or chemotherapy alone, P/D, and EPP were 11.7 (95% CI, 10.5–12.5), 20.5 (95% CI, 18.2–23.1), and 18.8 (95% CI, 17.2–20.9) months, respectively. The 30-day mortality was 2.6% after P/D and 4.1% after EPP (p = 0.401). According to multivariate analysis (n = 1227), age less than 70, epithelial histology, and chemotherapy were independent favorable prognostic factors. In the subset of 313 patients (25.5%) with all favorable prognostic factors, median survival was 18.6 months after medical therapy alone, 24.6 months after P/D, and 20.9 months after EPP (p = 0.596). Conclusions: Our data suggest that patients with good prognostic factors had a similar survival whether they received medical therapy only, P/D, or EPP. The modest benefit observed after surgery during medical treatment requires further investigation, and a large multicenter, randomized trial, testing P/D after induction chemotherapy versus chemotherapy alone in MPM patients with good prognostic factors, is needed.

Pulmonary lobectomy for lung cancer: a prospective study to compare patients with forced expiratory volume in 1 s more or less than 80% of predicted.

OBJECTIVE To compare post-operative course, lung function and survival of lung cancer patients with a forced expiratory volume in 1 s (FEV1) more or less than 80% of predicted submitted to lobectomy. METHODS The data of patients undergoing lobectomy for non small cell carcinoma at the Thoracic Surgery Unit of the Ospedale Maggiore Policlinico of Milan, Italy, were prospectively collected. Inclusion criteria were a radical resectable tumor with size less than 2.5 cm, negative mediastinal nodes, capability to complete pulmonary function tests, Exclusion criteria were FEV1 <40% of predicted, pre- or post-operative chemo or radiotherapy, lobe to be resected receiving more than 30% of the perfusion, incapacity to quit smoking. RESULTS Eighty-eight patients entered the study and were divided into two groups according to their FEV1%: 45 patients were included in control group (mean FEV1: 92.2%) and 42 in chronic obstructive pulmonary disease group (mean FEV1: 64.2%). Post-operative complications, operative mortality and actuarial survival were the same in the 2 groups. Six months after lobectomy, the mean changes in FEV1 were -14.9% for first group and -3.2% for second group (P<0.001). CONCLUSION Lobectomy for cancer can be performed successfully also in selected patients with chronic obstructive pulmonary disease. Post-operative course and survival of these patients is not different from that of patients with normal FEV1, on the contrary, patients with low FEV1 may lose less pulmonary function or even mend it.

Bridge to Lung Transplantation by Venovenous Extracorporeal Membrane Oxygenation: A Lesson Learned on the First Four Cases

Extracorporeal membrane oxygenation (ECMO) is the only therapeutic option for patients with ventilation-refractory hypercapnia while awaiting lung transplantation. Moreover, there is increasing success using ECMO for definitive respiratory failure in formerly healthy patients. This report describes the use of membrane oxygenation as a bridge to lung transplantation in 2 patients on the waiting list and in 2 previously healthy patients. Our experience showed that coagulation management, critical illness myopathy, and psychological disorders were the most critical problems. One patient died at 2 days after transplantation, 1 at 3 months, and 2 returned to their pretransplantation activities. We concluded that ECMO is an adequate bridge to lung transplantation but, especially in formerly healthy patients, an awake procedure is advisable for a successful outcome.

Identification of potential therapeutic targets in malignant mesothelioma using cell-cycle gene expression analysis.

Cell-cycle defects are responsible for cancer onset and growth. We studied the expression profile of 60 genes involved in cell cycle in a series of malignant mesotheliomas (MMs), normal pleural tissues, and MM cell cultures using a quantitative polymerase chain reaction-based, low-density array. Nine genes were significantly deregulated in MMs compared with normal controls. Seven genes were overexpressed in MMs, including the following: CDKN2C, cdc6, cyclin H, cyclin B1, CDC2, FoxM1, and Chk1, whereas Ube1L and cyclin D2 were underexpressed. Chk1 is a principal mediator of cell-cycle checkpoints in response to genotoxic stress. We confirmed the overexpression of Chk1 in an independent set of 87 MMs by immunohistochemistry using tissue microarrays. To determine whether Chk1 down-regulation would affect cell-cycle control and cell survival, we transfected either control or Chk1 siRNA into two mesothelioma cell lines and a nontumorigenic (Met5a) cell line. Results showed that Chk1 knockdown increased the apoptotic fraction of MM cells and induced an S phase block in Met5a cells. Furthermore, Chk1 silencing sensitized p53-null MM cells to both an S phase block and apoptosis in the presence of doxorubicin. Our results indicate that cell-cycle gene expression analysis by quantitative polymerase chain reaction can identify potential targets for novel therapies. Chk1 knockdown could provide a novel therapeutic approach to arrest cell-cycle progression in MM cells, thus increasing the rate of cell death.

FGFR4 Gly388Arg polymorphism may affect the clinical stage of patients with lung cancer by modulating the transcriptional profile of normal lung

The association of the fibroblast growth factor receptor 4 (FGFR4) Gly388Arg polymorphism with clinical stage and overall survival in a series of 541 Italian lung adenocarcinoma (ADCA) patients indicated a significantly decreased survival in patients carrying the rare Arg388 allele as compared to that in Gly/Gly homozygous patients [hazard ratio (HR) = 1.5; 95% confidence interval (CI) 1.1–1.9], with the decrease related to the association of the same polymorphism with clinical stage (HR = 1.8, 95% CI 1.3–2.6). By contrast, no significant association was detected in small series of either Norwegian lung ADCA patients or Italian lung squamous cell carcinoma (SQCC) patients. Single nucleotide polymorphisms of known FGFR4 ligands expressed in lung (FGF9, FGF18 and FGF19) were not associated with clinical stage or survival and showed no interaction with FGFR4. Analysis of gene expression profile in normal lungs according to FGFR4 genotype indicated a specific transcript pattern associated with the allele carrier status, suggesting a functional role for the FGFR4 polymorphism already detectable in normal lung. These findings confirm the significant association of the FGFR4 Gly388Arg polymorphism with clinical stage and overall survival in an Italian lung ADCA population and demonstrate a FGFR4 genotype‐dependent transcriptional profile present in normal lung tissue.

Ex vivo lung perfusion to improve donor lung function and increase the number of organs available for transplantation

This paper describes the initial clinical experience of ex vivo lung perfusion (EVLP) at the Fondazione Ca’ Granda in Milan between January 2011 and May 2013. EVLP was considered if donor PaO2/FiO2 was below 300 mmHg or if lung function was doubtful. Donors with massive lung contusion, aspiration, purulent secretions, pneumonia, or sepsis were excluded. EVLP was run with a low‐flow, open atrium and low hematocrit technique. Thirty‐five lung transplants from brain death donors were performed, seven of which after EVLP. EVLP donors were older (54 ± 9 years vs. 40 ± 15 years, EVLP versus Standard, P < 0.05), had lower PaO2/FiO2 (264 ± 78 mmHg vs. 453 ± 119 mmHg, P < 0.05), and more chest X‐ray abnormalities (P < 0.05). EVLP recipients were more often admitted to intensive care unit as urgent cases (57% vs. 18%, P = 0.05); lung allocation score at transplantation was higher (79 [40–84] vs. 39 [36–46], P < 0.05). After transplantation, primary graft dysfunction (PGD72 grade 3, 32% vs. 28%, EVLP versus Standard, P = 1), mortality at 30 days (0% vs. 0%, P = 1), and overall survival (71% vs. 86%, EVLP versus Standard P = 0.27) were not different between groups. EVLP enabled a 20% increase in available donor organs and resulted in successful transplants with lungs that would have otherwise been rejected (ClinicalTrials.gov number: NCT01967953).

Extracorporeal Lung Perfusion and Ventilation to Improve Donor Lung Function and Increase the Number of Organs Available for Transplantation

INTRODUCTION Ex vivo lung perfusion (EVLP) has been validated as a valuable technique to increase the pool of organs available for lung transplantation. MATERIAL AND METHODS After a preclinical experience, we obtained permission from the Ethics Committee of our institution to transplant lungs after EVLP reconditioning. ABO compatibility, size match, and donor arterial oxygen pressure (PaO(2))/fraction of inspired oxygen (FiO(2)) ≤ 300 mm Hg were considered to be inclusion criteria, whereas the presence of chest trauma and lung contusion, evidence of gastric content aspiration, pneumonia, sepsis, or systemic disease were exclusion criteria. We only considered subjects on an extra corporeal membrane oxygenation (ECMO) bridge to transplantation with rapid functional deterioration. Using Steen solution with packed red blood cells oxygenated with 21% O(2), 5% to 7% CO(2) was delivered, targeted with a blood flow of approximately 40% predicted cardiac output. Once normothermic, the lungs were ventilated with a tidal volume of 7 mL/kg a PEEP of 5 cmH(2)O and a respiratory rate of 7 bpm. Lungs were considered to be suitable for transplantation if well oxygenated [P(v-a) O(2) > 350 mm Hg on FiO(2) 100%], in the absence of deterioration of pulmonary vascular resistance and lung mechanics over the perfusion time. RESULTS From March to September 2011, six lung transplantations were performed, including two with EVLP. The functional outcomes were similar between groups: at T72 posttransplantation, the median PaO(2)/FiO(2) were 306 mm Hg (range, 282 to 331 mm Hg) and 323 mm Hg (range, 270 to 396 mm Hg) (P = 1, EVLP versus conventional). Intensive care unit ICU and hospital length of stay were similar (P = .533 and P = .663, respectively) with no mortality at 60 days in both groups. EVLP donors were older (49 ± 6 y versus 21 ± 7 y, P < .05), less well oxygenated (184 ± 6 mm Hg versus 570 ± 30, P < .05), displaying higher Oto scores (9.5 ± 0.7 versus 1.7 ± 1.5, P < .05). CONCLUSIONS The first 6 months of the EVLP program allowed us to increase the number of organs available for transplantation with short-term outcomes comparable to conventional transplantations.

Association of lung adenocarcinoma clinical stage with gene expression pattern in noninvolved lung tissue.

Associations between clinical outcome of cancer patients and the gene expression signature in primary tumors at time of diagnosis have been reported. To test whether gene expression patterns in noninvolved lung tissue might correlate with clinical stage in lung adenocarcinoma (ADCA) patients, we compared the transcriptome of noninvolved lung samples from 60 ADCA smoker patients of clinical stage I versus 60 patients with stage >I. Quantitative PCR of 10 genes with the most significant differential expression confirmed the statistical association with clinical stage in eight genes, six of which were downregulated in high‐stage patients. Five of these six genes were also downregulated in lung ADCA tissue as compared to noninvolved tissue. Studies in vitro indicated that four of the genes (SLC14A1, SMAD6, TMEM100 and TXNIP) inhibited colony formation of lung cancer cell lines transfected to overexpress the genes, suggesting their potential tumor‐suppressor activity. Our findings suggest that individual variations in the transcriptional profile of noninvolved lung tissue may reflect the lung ADCA patients predisposition to tumor aggressiveness.

β-Adrenergic agonist infusion during extracorporeal lung perfusion: Effects on glucose concentration in the perfusion fluid and on lung function

BACKGROUND We recently showed in a pig model of ex vivo lung perfusion (EVLP) that lung edema correlates with glucose consumption. We investigated whether salbutamol, a β-adrenergic receptor agonist known to upregulate fluid transport in the lung, modulates glucose concentration in the perfusate during EVLP. METHODS Lungs from domestic pigs underwent normothermic EVLP. At the end of controlled reperfusion, lungs were ventilated and perfused for 60 minutes, then randomized to salbutamol (β-Agonist) infusion or placebo (Control) for 180 minutes. Functional parameters were assessed. RESULTS In the β-Agonist group, glucose concentration decreased over time more than corresponding Control values (analysis of variance [ANOVA], p = 0.05). Mean pulmonary artery pressure (mPAP) was 16 ± 1 mm Hg in the β-Agonist group vs 21 ± 1 mm Hg in the Controls (ANOVA p < 0.05). Baseline mPAP was correlated with the drop of mPAP after the β-agonist infusion (R(2) = 0.856, p < 0.05). Dynamic compliance dropped from 51 ± 10 to 31 ± 6 ml/cm H(2)O in the β-Agonist group and from 60 ± 4 to 21 ± 3 ml/cm H(2)O in the Control group (ANOVA, p < 0.05 β-agonist vs Control). The Δ partial pressure of oxygen/fraction of inspired oxygen was 418 ± 15 and 393 ± 12 mm Hg in the β-Agonist and Control groups, respectively (t-test p = 0.106). CONCLUSIONS Glucose concentration in the perfusate was affected by salbutamol. Salbutamol was associated with lower pulmonary pressures and better lung mechanics. These data suggest a possible role for salbutamol as a pharmacologic adjunct during EVLP before transplantation.