Background: Surgical resection is the primary treatment for early-stage lung cancer, but little is known about the outcomes that truly matter to patients. This aim of our study was to identify the aspects of postoperative outcomes that matter most to patients undergoing lung cancer surgery and explore the influence of clinical and demographic factors on their importance ratings. Methods: We performed a cross-sectional study of patients undergoing lung resection for non-small cell lung cancer at our institution from November 2021 to May 2022. Patients were surveyed using a self- developed questionnaire and the European Organisation for Research and Treatment of Cancer core health- related quality of life questionnaire (EORTC QLQ-C30) prior to surgery. Ordinal logistic regression was performed to determine associations between individual patient factors and outcome importance ratings. Results: Forty patients completed the survey during the study period. Patients prioritized oncologic outcomes, with 95% rating R0 resection and cancer recurrence as "very important". Other important factors included overall survival (90%), postoperative complications (e.g., myocardial infarction: 92.5%, infection: 87.5%), and the need for reoperation (82.5%). Health-related quality of life factors, such as chronic pain (77.5%) and the ability to return to normal physical and exercise levels (75%), were also highly valued. Certain patient clinical and demographic factors demonstrated significant associations with importance placed on certain outcomes. Preoperative health-related quality of life scores did not influence outcome importance ratings. Conclusions: This study provides insights into the outcomes that matter most to patients undergoing lung cancer surgery. Oncologic outcomes and postoperative complications were prioritized, while scar- related factors were less important. Patient preferences varied based on demographic and clinical factors. Understanding these preferences can enhance shared decision-making and improve patient-centered care in thoracic surgical oncology.

Objective: Surgical mortality has traditionally been assessed at arbitrary intervals out to 1 year, without an agreed optimum time point. The aim of our study was to investigate the time-varying risk of death after lobectomy to determine the optimum period to evaluate surgical mortality rate after lobectomy for lung cancer. Methods: We performed a retrospective study of patients undergoing lobectomy for lung cancer at our institution from 2015 to 2022. Parametric survival models were assessed and compared with a nonparametric kernel estimate. The hazard function was plotted over time according to the best-fit statistical distribution. The time points at which instantaneous hazard rate peaked and stabilized in the 1-year period after surgery were then determined. Results: During the study period, 2284 patients underwent lobectomy for lung cancer. Cumulative mortality at 30, 90, and 180 days was 1.3%, 2.9%, and 4.9%, respectively. Log-logistic distribution showed the best fit compared with other statistical distribution, indicated by the lowest Akaike information criteria value. The instantaneous hazard rate was greatest during the immediate postoperative period (0.129; 95% confidence interval, 0.087-0.183) and diminishes rapidly within the first 30 days after surgery. Instantaneous hazard rate continued to decrease past 90 days and stabilized only at approximately 180 days. Conclusions: In-hospital mortality is the optimal follow-up period that captures the early-phase hazard during the immediate postoperative period after lobectomy. Thirty-day mortality is not synonymous to "early mortality," as instantaneous hazard rate remains elevated well past the 90-day time point and only stabilizes at approximately 180 days after lobectomy. (JTCVS Open 2023;16:931-7)

Objectives: Advances in perioperative management for thoracic surgery have accelerated the postoperative recovery of patients by decreasing postoperative pain and the incidence of complications. We aimed to study whether it's safe to remove chest drains on table in selected cases. Methods: This was a 5-year retrospective analysis of protocolized chest-drain removal on the operating table. The chest drain was removed in patients undergoing sublobar/wedge lung resection and other minor thoracic procedure (pleural biopsy, mediastinal mass biopsy/resection) via a thoracoscopic approach (video-assisted thoracoscopic surgery). Chest drains were removed at the end of the operation if air leak as documented by the digital drain was less than 20 mL/min. Outcome data on postdrain removal pneumothorax, effusion, and need for further intervention were obtained by reviewing the postoperative chest films, all reported by a radiologist. Results: Between 2016 and 2021, 107 patients underwent drain removal in theater. Mean age (standard deviation) was 58 (17) years and 54 (50.5%) were male. Postdrain removal pneumothorax occurred in 22 patients (21%), pleural effusion in 6 (5.6%), and 21 of 22 postoperative pneumothoraces were managed conservatively without reinsertion of chest drain. As it is our standard policy to leave no pneumothorax in patients undergoing surgical management of primary spontaneous pneumothorax, only 1 such patient (0.9%) had a drain reinserted as a result. The median (interquartile) length of hospital stay was 1 day (1-2), and 14 patients (13%) were discharged on surgery day. Conclusions: Our results demonstrate that on table chest-drain removal in selected cases is safe and repeatable using a digital drain, challenging the practice of routine drain insertion after thoracic surgery.

Introduction Ward rounds are vital clinical processes that facilitate an opportunity for daily review and management of thoracic surgery inpatients. The aim of this study was to compare thoracic surgery ward round documentation against locally agreed standards and design a template to improve the detail and uniformity of this process to enhance patient care. Materials and methods Data for this audit was collected retrospectively and prospectively. Data was collected during three auditing periods and managed on Microsoft Excel. Descriptive statistics were used for its analysis. Chi-square and Fisher’s Exact tests were used to test for differences in reporting rates. Results and discussion Initially, a total of 199 ward round notes were reviewed. Imaging results (19%) and discharge planning (23%) were not reported. eCARE (electronic Clinical Assessment for Round Evaluation) was developed to ensure that all aspects of patient evaluation recommended by the guidelines were included. Reporting rates significantly improved after such changes. We analysed the effect of the new ward round note on discharge planning (23.3 vs 41%, p<0.001), complication rates (32.6 vs 21.9%, p=0.03), post-surgical length of stay (LOS) (7.0 vs 5.0, p<0.001). Conclusion Over a year, we audited the Thoracic Surgery Department's ward round documentation against locally agreed standards in line with national recommendations. Several important items were not regularly reported. Using closed-ended questions improved reporting rates, and patient care was optimised. Further research should explore the impact of this new documentation method on patient care and postoperative outcomes in our Trust as well as other cardiothoracic centres.

Background: Chest drain management is a variable aspect of postoperative care in thoracic surgery, with different opinion for air and drain volume output. We aim to study if acceptable safety was maintained using air leak criteria alone.Methods: A 9-year retrospective analysis of protocolised chest drain management using digital drain air leak cut off less than 20 mL/min for more than 6 h for drain removal in patients undergoing general thoracic surgery. We excluded patients if a chest drain was not required nor removed during admission or if patients underwent volume reduction or pneumonectomy. Withdrawal criteria were suspected bleeding or chylothorax. Postoperative films were reviewed to document post-drain removal pneumothorax, pleural effusion, and reintervention (drain re-insertion).Results: Between 2012 and 2021, 1,187 patients had thoracic surgery under a single surgeon. Following exclusion and withdrawal criteria, 797 patients were left for analysis. The mean age [standard deviation (SD)] was 61 [16] years and 383 (48%) were male. Median [interquartile range (IQR)] duration of drain insertion was 1 [1-2] day with a median length of hospital stay of 4 [2-6] days. Post-drain removal pneumothorax was observed in 141 (17.7%), post-drain removal pleural effusion was observed in 75 (9.4%) and re-intervention (reinsertion of chest drain) required in 17 (2.1%). Conclusions: Our results demonstrate acceptable levels of safety using digital assessment of air leak as the sole criteria for drain removal in selected patients after general thoracic surgery.

OBJECTIVES: We sought to evaluate the impact of different national clinical guidelines (with consistent and conflicting recommendations) on clinician's practice in the UK. METHODS: In this cohort study, we analysed data from National Lung Cancer Audit comprising all patients diagnosed with lung cancer between 2008 and 2013 within England and Wales for consistent (British Thoracic Society and National Institute of Clinical Excellence) recommendations for lower/more permissive lung function but opposing stage (N2) selection parameters for surgery. RESULTS: From 2008 to 2013, data from 167 192 patients with primary lung cancers were included. The proportion of patients undergoing surgery for lung cancer increased from 9.5% to 20.5% in 2013 (P < 0.001) as the number of general thoracic surgeons in the UK increased from 40 to 81 in the corresponding timeframe. Mean forced expiratory volume in 1 s of surgical patients increased from 76% (22) to 81% (22) in 2013 (P < 0.001). Of the patients undergoing surgery, the proportion of patients with N2 disease across the 6-year interval was broadly consistent between 8% and 11% without any evidence of trend (P = 0.125). CONCLUSIONS: Within 3 years of new clinical guideline recommendations, we did not observe any overall change suggesting greater selection for surgery on lower levels of lung function. When presented with conflicting recommendation, no observable change in selection was noted on surgery for N2 disease. The observed increase in surgical resection rates is more likely due to greater access to surgery (by increasing number of surgeons) rather than any impact of guideline recommendations.

Ischaemia-reperfusion injury (IRI) encompasses the deleterious effects on cellular function and survival that result from the restoration of organ perfusion. Despite their unique tolerance to ischaemia and hypoxia, afforded by their dual (pulmonary and bronchial) circulation as well as direct oxygen diffusion from the airways, lungs are particularly susceptible to IRI (LIRI). LIRI may be observed in a variety of clinical settings, including lung transplantation, lung resections, cardiopulmonary bypass during cardiac surgery, aortic cross-clamping for abdominal aortic aneurysm repair, as well as tourniquet application for orthopaedic operations. It is a diagnosis of exclusion, manifesting clinically as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Ischaemic conditioning (IC) signifies the original paradigm of treating IRI. It entails the application of short, non-lethal ischemia and reperfusion manoeuvres to an organ, tissue, or arterial territory, which activates mechanisms that reduce IRI. Interestingly, there is accumulating experimental and preliminary clinical evidence that IC may ameliorate LIRI in various pathophysiological contexts. Considering the detrimental effects of LIRI, ranging from ALI following lung resections to primary graft dysfunction (PGD) after lung transplantation, the association of these entities with adverse outcomes, as well as the paucity of protective or therapeutic interventions, IC holds promise as a safe and effective strategy to protect the lung. This article aims to provide a narrative review of the existing experimental and clinical evidence regarding the effects of IC on LIRI and prompt further investigation to refine its clinical application.

Isotretinoin is a retinoid and derivative of vitamin A used to treat severe recalcitrant acne vulgaris. By affecting the cell cycle, cell differentiation, survival, and apoptosis it reduces sebum production, prevents the blockage of pores, and growth of acne-causing bacteria. Additionally, isotretinoin may interact with FoxO1, which may explain a substantial number of isotretinoin's unexplained actions.

OBJECTIVES:Unplanned readmission is defined as the return to inpatient hospitalization within 30 days after discharge. Worldwide, its incidence after lung resection ranges between 8% and 50%, and it has been shown to impact both patient recovery and healthcare resources. Our goal was to identify the risk factors to prioritize early follow-ups. METHODS:We analysed data from the database of the Grupo Español de Cirugía Torácica Video-Asistida from 33 thoracic surgery departments over 15 months. Standard tests were used to compare the different risk groups. Our goal was to present the most relevant explanatory variables for readmission. RESULTS:A total of 174 of 2808 patients (6%) underwent unplanned readmission after a lobectomy. Of all the preoperative individual characteristics, only lung function was found to be a risk factor for readmission [forced expiratory volume in 1 s < 80%, risk ratio (RR) 1.78, P < 0.001; diffusing capacity of carbon monoxide <60%, RR 1.6, P = 0.02; and VO2 < 20 ml/kg/min, RR 1.59, P = 0.02]. The tumour's characteristics and the stage of the disease did not have an influence on the readmission rates. In the readmitted cohort, an open approach or thoracotomy was associated with more frequent readmissions (RR 1.77; P < 0.001). Strong adhesions (RR 1.81; P < 0.001) or adhesions occupying more than half of the hemithorax (RR 1.73, P < 0.001) were also found to be risk factors for readmission and for longer operative times. A length of stay of >10 days after a lobectomy was found to be a risk factor for readmission (RR 1.9). CONCLUSIONS:We identified preoperative, intraoperative and postoperative risk factors for readmission. This information can be a useful tool to help with the prioritization of early follow-ups, especially in centres with high workloads.

A 77-year-old woman with multiple ground-glass opacities, the largest of which measured 21 mm, has a biopsy-proven primary lung adenocarcinoma in her right upper lobe. We performed a 3-port right-sided VATS using the Copenhagen approach. There was no pleural effusion or evidence of pleural metastatic spread. A tumor was identified in the upper lobe. The surrounding lung tissue appeared normal.  We performed a multilevel intercostal block using 0.25% levobupivacaine. The inferior pulmonary ligament was divided. The superior pulmonary vein and 2 branches of the pulmonary artery to the right upper lobe were dissected, encircled, and divided using tan reloads of the Endo GIA stapler. The right upper lobe bronchus was dissected, encircled, and divided in a similar fashion using a purple reload of the Endo GIA stapler following a successful test inflation of the lower and middle lobes. The horizontal fissure was completed with further firings of the stapler. Lymph nodes from stations 2, 4, 7, 8, 9, 10, and 11 were sampled and sent separately for histological analysis. There was no parenchymal or stump leak to 20 cm H20 on the test inflation. Hemostasis and pneumostasis were checked and ensured. A single 24 Fr drain was placed in the apex. Hemostasis was complete. The incision was closed in layers.

Background: Over the last years, our knowledge on pathogenesis of gastric MALT lymphoma has greatly improved, but its morphological diagnosis is still hampered by overlapping histological features with advanced chronic gastritis. MicroRNAs are deregulated in lymphomas, but their role and usefulness in gastric MALT lymphoma has not been extensively investigated. Materials and Methods: We analyzed the expression of 384 miRNAs using TaqMan microRNA assay in a training series of 10 gastric MALT lymphomas, 3 chronic gastritis and 2 reactive lymph nodes. Then, significantly deregulated miRNAs were individually assessed by real-time PCR in a validation series of 16 gastric MALT lymphomas and 12 chronic gastritis. Results: Gastric MALT lymphoma is characterized by a specific miRNA expression profile. Among the differentially expressed miRNAs, a significant overexpression of miR-142-3p and miR-155 and down-regulation of miR-203 was observed in gastric MALT lymphoma when compared to chronic gastritis. Conclusion: miR-142-3p, miR-155 and miR-203 expression levels might be helpful biomarkers for the differential diagnosis between gastric MALT lymphomas and chronic gastritis.

Abstract Background MicroRNAs (miRNAs) act as negative regulators of expression of genes that participate in cellular proliferation, apoptosis and/or carcinogenesis. MiRNAs have been shown to play a role in both solid and hematological tumors. Interestingly, several miRNA profiles have been differentially related to the mutational status of multiple tumors. A mutation in the JAK2 kinase (V617F) has been associated with essential thrombocythemia (ET) in around 40–50% of cases. However, the biological basis of ET in patients lacking the JAK2V617F mutation is still unknown. miRNAs may be crucial to the development of ET in these patients, yet their role in JAK2V617F-negative ET remains to be elucidated. Aim To characterize the expression pattern of miRNAs involved in JAK2V617F-negative ET and to identify the targets of these miRNAs. Methods Total RNA was extracted from isolated platelets from 19 ET patients (10 JAK2V617F-positive and 9 JAK2V617F-negative) and 10 healthy controls. The expression of 670 mature miRNAs was determined by TaqMan Human MicroRNA Arrays v2.0 (Applied Biosystems) in an ABI 7900 HT real time PCR system. miRNA expression data was analyzed by the 2-ΔΔCt method, using RNU48 as endogenous control. Statistical analyses were performed with TiGR MultiExperiment Viewer and R software v 2.13.To identify molecular pathways potentially altered by the expression of multiple miRNAs, we used Diana-mirPath, which performs an enrichment analysis of multiple miRNA target genes, comparing each set of miRNA targets to all known KEGG pathways. The mRNA expression of identified putative targets was analyzed by TaqMan gene expression assays (Applied Biosystems) and correlated with miRNA expression in order to select candidate genes for further target validation by Renilla-Luciferase assay and Western Blot. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in K562 together with 1μM of modified psicheck2 vector containing the 3’UTR region to be validated. Renilla luciferase levels were measured at 24h post-tranfection. The miRNAs identified in the Renilla-Luciferase assay were further confirmed by Western Blot. 100nM of pre-miR/pre-miR negative control were transfected in the HL60 cell line and protein levels were measured at 24h post-transfection. Anti-SOCS1 (ab119954) and Anti-SOCS3 (ab62584) primary antibody (abcam) were used. Results Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic miRNA signature (p<0.0001). ET patients harbored a distinctive signature of 101 miRNAs, 99 of which were downregulated. Furthermore, a 40-miRNA signature was found to be differentially expressed between JAK2V617F-positive and JAK2V617F-negative ET patients. Diana-mirPath analysis showed that 8 of these 40 miRNAs had putative targets in the JAK-STAT pathway. The mRNA expression analysis of these genes showed an inverse correlation between the expression of three miRNAs and their target genes. An inverse correlation was found between SOCS1 and miR-221 (r2 = -0.719, p=0.001), SOCS3 and miR-221 (r2 = -0.644, p=0.005), SOCS3 and miR-203 (r2 = -0.447, p=0.072) and PTPN11 and miR-23a (r2 = -0.494, p=0.044). All three miRNAs were upregulated in JAK2V617F-negative ET patients in comparison with JAK2V617F-positive ET patients. The Renilla-Luciferase assay validated SOCS1 as a target of miR-221 (28.9% Renilla luciferase protein reduction, p=0.002) and SOCS3 as a target of miR-203 (19.6% Renilla luciferase protein reduction, p=0.04). No significant modifications were observed for PTPN11. SOCS1 and SOCS3 were further validated by Western Blot, with significant reduction of protein levels of SOCS1 (16%) and SOCS3 (19%) after increasing the levels of miR-221 and miR-203 respectivelly. Conclusion A 40-miRNA signature is differentially expressed in JAK2V617F-negative and JAK2V617F-positive ET patients. Two of these miRNAs, miR-221 and miR-203, target SOCS1 and SOCS3, which are involved in the JAK-STAT pathway. Further investigation is warranted to shed light on the role of these miRNAs in the development of JAK2V617F-negative ET. Acknowledgments This study was supported in part by grants EC10-136, FISPI10/01807, RD09/0076/00036, RD12/0036/0010, 2009SGR929 and AECC Catalunya 2011 Disclosures: No relevant conflicts of interest to declare.

The Fc gamma receptor IIb (FcϒRIIb) is an inhibitory receptor that plays a central role in limiting B-cell activation by inhibiting B-cell receptor (BCR) signaling. To date, no studies have evaluated and compared the role of FcϒRIIb function in normal and chronic lymphocytic leukemia (CLL) human B cells. Using a specific anti-human FcϒRIIb monoclonal antibody (MacroGenics, Inc.), we previously found that FcϒRIIb expression on 84 CLL clones was significantly reduced compared with that on normal human B cells from 24 age- and sex-matched controls; this was consistent with the activated nature of CLL cells. Interestingly, we also observed that the expression of FcϒRIIb varied within individual CLL clones, being higher in CD38+ and CD49d+ cells than in CD38– and CD49d– leukemic cells. In this study, we investigated whether engagement of FcϒRIIb on CLL cells inhibited BCR signaling and, hence, controlled B-cell growth as for normal B lymphocytes. To address this, we used 2 forms of the same polyclonal rabbit antibody (pAb) preparation to human immunoglobulin (Ig)M to crosslink the BCR: whole antibodies and their pepsin-digested F(ab’)2 fragments. The former molecules simultaneously crosslink BCRs and FcϒRIIb via the Fab and Fc domains, respectively, while the latter crosslink BCRs only because they lack Fc domains. Purified B cells from 14 CLL patients and 9 healthy subjects were co-cultured with a fibroblast cell line (HS-5), interleukin-4 (20 ng/mL), and 10 mg/mL of either F(ab’)2 fragments or whole pAbs. After 48 and 72 hours, B cells were (1) labeled with Annexin V and TO-PRO®-3 (Invitrogen) to assess cell viability; (2) stained with monoclonal antibodies to CD69 to determine cell activation; and (3) exposed to 5-ethynyl-2’-deoxyuridine to measure cell proliferation. Each of these three parameters was analyzed using flow cytometry. In normal B cells, significant and equal increases in cell activation and viability were observed in response to stimulation with either the F(ab’)2 fragment or whole form of pAbs. However, the ability to induce cell proliferation differed, with F(ab’)2 fragments leading to more proliferation than cells stimulated with the whole antibody (p = 0.004), consistent with an FcϒRIIb-mediated inhibition of BCR-induced proliferation. For CLL cells, however, responses to F(ab’)2 fragments and the whole pAbs differed from those of normal B lymphocytes for some readouts. After BCR crosslinking with F(ab’)2 fragments, the median percentage of activated and viable CLL cells increased from 48.3% to 56.5% and from 25% to 40.5% (p = 0.033 and p = 0.002, respectively), as for normal B cells. However, after BCR crosslinking with whole pAbs, which colligate both the BCR and FcϒRIIb, cell activation and viability was increased to an even greater extent than with the digested form (from 48% to 59%, p < 0.001, and from 25% to 46%, p = 0.002, respectively). Although CLL cells did not proliferate as well as normal B cells after stimulation with either whole or F(ab’)2 fragment antibodies, both forms of the anti-IgM pAbs induced cell proliferation to equal degrees. In addition, surprisingly, greater FcϒRIIb expression on CLL cells led to greater levels of proliferation. Therefore, we investigated further the extent that differences in response to F(ab’)2 fragments and whole antibody were due to differences in the expression of FcϒRIIb within the CLL clones. In 4 samples investigated up to the time of abstract submission, whereas greater levels of activation (i.e. CD69 expression) were detected on CD38+ cells prestimulation, no differences were observed in activation nor in viability between CD38+ and CD38– cells after stimulation with whole or F(ab’)2 fragment antibodies. Interestingly, CD38– cells stimulated with F(ab’)2 fragments showed a lower proliferation level than CD38– cells after stimulation with the whole pAbs that simultaneously crosslink BCR and FcϒRIIb. These differences were not observed in CD38+ cells. These findings demonstrate that simultaneous crosslinking of BCR and FcϒRIIb on CLL B cells does not diminish B-cell proliferation, in contrast to what is observed in normal B cells. This suggests that the ability of FcϒRIIb to inhibit BCR signaling is attenuated in patients with CLL and might actually enhance responsiveness to BCR-mediated signals. This hypothesis is supported by the relationship between FcϒRIIb expression on CLL cells and functional responses to signals simultaneously delivered by BCR and FcϒRIIb. The importance of this difference on CLL cell survival and proliferation in vivo and its correlation with patients' outcomes should be explored.

Abstract Abstract 3912 Poster Board III-848 Background The myeloproliferative neoplasms (MPNs) include different diseases presenting several mutations in variable frequency. The JAK2V617F mutation is present in 90-95% Polycythemia Vera (PV), 50-60% Essential Thrombocythemia (ET) and 50-60% Primary Myelofibrosis (PMF) patients. In addition, JAK2 exon 12 mutations are observed in 1-3% of PV patients and mutations in the thrombopoietin receptor gene (c-MPL) (S505N, W515K/L) are present in a 5-9% of PMF and in a 1-4% of ET patients. Recently, acquired mutations of the Ten-Eleven Translocation (TET) 2 gene, have been reported in about 14% of sporadic MPNs. TET2 mutations may precede the acquisition of JAK2V617F predisposing to a MPN development. However, the incidence of TET2 mutations in patients lacking the JAK2V617F mutation has not been extensively studied. Aim To analyze the incidence of TET2 mutations in a cohort of MPN patients negative for JAK2V617F, JAK2 exon 12 mutations and c-MPL exon 10 mutations (S505N or W515K/L). Patients and methods From a whole cohort of 241 patients with MPN (93 PV, 16 PMF and 132 ET) we excluded those patients positive for JAK2V617F (determined by quantitative allele-specific PCR), JAK2 exon 12 mutations or c-MPL exon 10 mutations (S505N or W515K/L) (analyzed by direct sequencing). We analyzed the TET2 gene in 5 PV, 5 PMF and 53 ET patients lacking any of the aforementioned molecular markers. The mutational analysis of the coding sequence of TET2 was performed by direct sequencing using cDNA from granulocytes. In 13 patients, DNA from T lymphocytes was obtained to indentify the presence of single nucleotide polymorphisms (SNPs) in germline DNA. Results Sixty-three patients were screened for mutations in the whole coding sequence of the TET2 gene. Only 3 ET patients (4.7%) presented deleterious mutations in the TET2 gene. The three distinct TET2 mutations were: Q706X, S1848X and V1395_R1400delinsR. In 48 out of 63 (76.1%) patients we observed a total of 13 different missense mutations and 2 silent mutations in the coding sequence of the gene. The most frequent missense mutation was the I1762V which was detected in 27 patients. In 13 patients whose matched normal DNA was available, we analyzed the presence of missense mutations being all of them present in the control DNA suggesting that they were SNPs and not acquired mutations. The two nonsense mutations (Q706X and S1848X), were not present in matched normal tissue indicating that these mutations were somatically acquired in myeloid cells. Conclusions TET2 pathogenic mutations are infrequent (<5%) in myeloproliferative neoplasms negative for JAKV617F, JAK2 exon 12 and c-MPL (S505, W515K/L) gene mutations. The role and the biological significance of missense mutations in the coding sequence of TET2 has to be elucidated. Acknowledgments Fellowship FI2008 (AGAUR) to LMM-A, FIS EC07/90791 Disclosures: No relevant conflicts of interest to declare.

The behavior of classic Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the tumor cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. We examined the influence of our previously reported 25-microRNA signature for cHL on clinical outcome in 89 homogeneously treated cHL patients with a median follow-up of 80 months. Patients with low miR-135a expression had a higher probability of relapse (P = .04) and a shorter disease-free survival (P = .02). Functional analysis of cHL cell lines showed that mature miR-135a levels increased after pre-miR-135a transfection, causing apoptosis and decreased cell growth. Target analysis showed a direct regulation by miR-135a of JAK2, a cytoplasmic tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. miR-135a-mediated JAK2 down-regulation led to decreased mRNA and protein levels of the antiapoptotic gene Bcl-xL, suggesting a role for Bcl-xL in miR-135a/JAK2-mediated apoptosis. Our findings confirm the critical role of miR-135a in the survival of cHL cells and in the prognosis of cHL patients, indicating that novel treatment approaches targeting miR-135a may potentially benefit these patients.

MicroRNAs (miRNAs) are negative regulators of expression of genes involved in hematopoiesis. The present study sought to link hematopoiesis-relevant miRNAs with myelodysplastic syndromes (MDS) and MDS progression to acute myeloid leukemia (AML). We assessed 25 mature miRNAs in total RNA from bone marrow (BM) and peripheral blood (PB) of 25 newly diagnosed patients with MDS and 12 controls. Twelve miRNAs in BM and six in PB were differentially expressed between patients with MDS and controls. Three of these miRNAs, belonging to the cluster 17-92, were overexpressed in both BM and PB. miR-15a in BM ( p = 0.034) and miR-16 in PB ( p = 0.005) were differentially expressed between low-risk and high-risk groups. miR-222 ( p = 0.0023) and miR-181a ( p = 0.014) expression was higher in AML than in MDS in both BM and PB. This study adds further evidence to the role of miRNAs in the pathogenesis of MDS and their transformation into AML.

Abstract Abstract 1912 Poster Board I-935 Background: Essential Thrombocythemia (ET) and Polycythemia Vera (PV) are myeloproliferative neoplasms (MPN) arising from a multipotent hematopoietic stem cell characterized by an unregulated production of platelets, red cells and white cells alone or in combination, a tendency to clonal evolution and an increased risk of thrombohemorrhagic complications. MicroRNAs (miRNA) are negative regulators of genes involved in cellular proliferation, apoptosis and/or carcinogenesis. Aim: To analyze the expression pattern of miRNA between PV and ET patients and to find distinctive signatures in ET patients according to JAK2V617F and c-MPL mutational status. Material and Methods: Total RNA was extracted from peripheral blood granulocytes of 50 ET patients, 10 PV patients and 10 controls. Median age of patients was 57 years (range, 20-88); males 36%. The JAK2V617F mutation was present in 23 (46%) of 50 ET patients and in all PV patients. MPL mutations were present in 5 (18%) of 27 JAK2V617F negative cases (3 cases MPLW515L, 1 case MPLW515K and 1 case MPLS505N). miRNA expression was profiled in 384 miRNA via Taqman Low Density Array in ABI PRISM 7900. Expression data was normalized with RNU48 and relative quantification was calculated with the 2–σσCt method. The data were presented as log10 of relative quantity of target miRNA. Median of normal controls was used as calibrator for all samples. Data were analyzed by means of Significant Analysis of MicroArrays (SAM), Prediction Analysis of MicroArrays (PAM) and Class Comparison methods using BRB array tools version 3.7.0 and TIGR multiexperiment viewer version 4.3. Results: We found a general downregulation of miRNA in ET and PV patients respect to normal controls. A set of 29 miRNA allowed us to discriminate between ET and PV versus normal controls; three of these miRNA were up-regulated and 16 down-regulated in PV and ET vs. normal controls with a >2 fold change and p value <0.01. A distinctive signature of 79 miRNAs differentiated ET, PV and controls and a hierarchical clustering analysis defined miRNA expression profiles of the three particular groups. When we compared miRNA differentially expressed between PV and ET patients, we found nine miRNA, 4 up-regulated and 5 down-regulated in ET with respect to PV patients (p<0.01). Statistical comparisons between ET JAK2V617F-positive and ET JAK2V617F-negative cases showed a distinctive signature of 13 miRNA that allowed us to discriminate between the two groups. In addition, we also found in JAK2V617F cases 19 miRNA differentially expressed between MPL positive and MPL negative patients, with a >2 fold change and p<0.01. Finally, an increased expression of miR-142-5p correlated with JAK2V617F allele burden in ET patients Conclusions: Our study demonstrates that ET and PV can be defined by specific signatures of miRNA. In ET, some miRNA allow us to discriminate cases according to JAK2V617F mutational status and also between MPL-positive and MPL-negative JAK2V617F-negative patients. Research Funding: FIS EC07/90791 Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1235 Poster Board I-257 BAFF (B-cell activating factor) and APRIL (a proliferation-inducing ligand) are TNF family proteins that upregulate anti-apoptotic genes through the NF-kB pathway. Studies in vitro suggest that BAFF and APRIL protect neoplastic B cells from apoptosis in chronic lymphoproliferative disorders (CLPD) including chronic lymphocytic leukemia (CLL). Serum BAFF levels have been previously shown to be lower in CLL than in other CLPD or normal subjects. To contribute to a better understanding of their role in CLL, we analyzed BAFF and APRIL at mRNA and protein serum levels and their receptors [transmembrane activator and CAML interactor (TACI), B-cell maturation antigen (BCMA) and BAFF receptor (BAFF-R)] by flow cytometry, in 82 patients with CLL, 36 with other CLPD and 35 age- and sex-matched controls. mRNA BAFF and APRIL levels were calculated as a the percentage of expression referred to an internal control and the receptor expression as the ratio between the mean fluorescence intensity (MFI) of the receptor antibody and the MFI of the isotype control. Patients with CLL showed significantly lower median BAFF and APRIL levels (0.63 μg/ml and 3.18 μg/ml) than those with other CLPD (1.27 μg/ml and 5.51 μg/ml) (p<0.05). Moreover, BAFF but not APRIL was lower in CLL than in healthy subjects (0.63 μg/ml vs. 0.77 μg/ml; p<0.0001). Serum BAFF levels and blood lymphocyte counts were inversely correlated. Likewise, in follicular lymphoma patients who had circulating neoplastic B cells, median BAFF levels was 0.84 μg/ml vs. 1.46 μg/ml in those without detectable neoplastic cells in blood (p<0.05). We also examined the expression of BAFF and APRIL in purified CD19+ cells from 19 CLL patients and 10 healthy controls. All CLL and normal B cells expressed BAFF and APRIL although heterogeneously. Nevertheless, BAFF and APRIL were lower in CLL than in normal B cells (median: 6.24% and 12.73% in CLL vs. 11.54% and 42.26% in controls). In CLL, mRNA BAFF expression inversely correlated with BAFF serum levels. As far as BAFF and APRIL receptors are concerned, BAFF-R was the one most highly expressed in CLL and normal B cells (MFI ratios of 167.3 and 157.2, respectively). TACI and BCMA were also expressed in all CLL cells and normal B cells (MFI ratios TACI: 1.70 and 2.41; BCMA: 9.51 and 4.72, respectively), but at a significantly lower level than BAFF-R (p<0.001). Furthermore, whereas BCMA MFI ratio was significantly higher in CLL than in normal B cells (p<0.05), no differences were observed in the expression of TACI and BAFF-R. TACI expression was heterogenous in CLL cells. BAFF-R inversely correlated with BAFF and APRIL serum levels. From a clinical standpoint, there is some indication that BAFF and APRIL serum levels as well as their expression in CLL cells may correlate with clinical and biological characteristics of the disease. No significant relationship was observed between BAFF and APRIL and IGVH mutational status, ZAP-70, CD38 or cytogenetics. However, an inverse correlation was observed between BAFF serum levels and blood lymphocyte counts as well as advanced clinical stage (p<0.05). In contrast, APRIL serum levels were only correlated with CD38 expression, the higher the expression of CD38 the higher the APRIL. Although blood lymphocyte counts and BAFF serum levels are correlated, a multivariate analysis showed that these two variables along with poor risk cytogenetics were independent predictors of progression (poor risk cytogenetics RR=11.699, p<0.05; high blood lymphocyte count RR=9.780, p<0.05 and low serum BAFF RR= 6.098, p<0.05). In summary this study confirms that BAFF and APRIL serum levels are lower in CLL than in other CLPD. In patients with CLL, BAFF serum and mRNA levels correlate with blood lymphocyte count and advanced clinical stage but not with other well known prognostic factors. Finally, although BAFF correlates with blood lymphocyte counts, our results suggest that BAFF serum levels have independent prognostic significance. Disclosures No relevant conflicts of interest to declare.

MicroRNAs (miRNAs) are negative regulators of gene expression that play an important role in hematopoiesis and tumorigenesis. We analyzed miRNA expression in classic Hodgkin lymphoma (cHL) and the influence of Epstein-Barr virus (EBV) infection on the miRNA expression profiles. The expression of 157 miRNAs in lymph nodes from 49 cHL patients and 10 reactive lymph nodes (RLNs) was analyzed by real-time polymerase chain reaction (PCR). Hierarchic clustering revealed 3 well-defined groups: nodular sclerosis cHL, mixed cellularity cHL, and RLNs. A distinctive signature of 25 miRNAs differentiated cHL from RLNs, and 36 miRNAs were differentially expressed in the nodular sclerosis and mixed cellularity subtypes. These results were validated in a set of 30 cHLs and 5 RLNs, and in 3 cHL cell lines. miR-96, miR-128a, and miR-128b were selectively down-regulated in cHL with EBV. Our findings suggest that miRNAs play an important role in the biology of cHL and may be useful in developing therapies targeting miRNAs.