Luka Brcic

Nuclear grade and necrosis predict prognosis in malignant epithelioid pleural mesothelioma: a multi-institutional study

A recently described nuclear grading system predicted survival in patients with epithelioid malignant pleural mesothelioma. The current study was undertaken to validate the grading system and to identify additional prognostic factors. We analyzed cases of epithelioid malignant pleural mesothelioma from 17 institutions across the globe from 1998 to 2014. Nuclear grade was computed combining nuclear atypia and mitotic count into a grade of I–III using the published system. Nuclear grade was assessed by one pathologist for three institutions, the remaining were scored independently. The presence or absence of necrosis and predominant growth pattern were also evaluated. Two additional scoring systems were evaluated, one combining nuclear grade and necrosis and the other mitotic count and necrosis. Median overall survival was the primary endpoint. A total of 776 cases were identified including 301 (39%) nuclear grade I tumors, 354 (45%) grade II tumors and 121 (16%) grade III tumors. The overall survival was 16 months, and correlated independently with age (P=0.006), sex (0.015), necrosis (0.030), mitotic count (0.001), nuclear atypia (0.009), nuclear grade (<0.0001), and mitosis and necrosis score (<0.0001). The addition of necrosis to nuclear grade further stratified overall survival, allowing classification of epithelioid malignant pleural mesothelioma into four distinct prognostic groups: nuclear grade I tumors without necrosis (29 months), nuclear grade I tumors with necrosis and grade II tumors without necrosis (16 months), nuclear grade II tumors with necrosis (10 months) and nuclear grade III tumors (8 months). The mitosis–necrosis score stratified patients by survival, but not as well as the combination of necrosis and nuclear grade. This study confirms that nuclear grade predicts survival in epithelioid malignant pleural mesothelioma, identifies necrosis as factor that further stratifies overall survival, and validates the grading system across multiple institutions and among both biopsy and resection specimens. An alternative scoring system, the mitosis–necrosis score is also proposed.

Primary patient-derived lung adenocarcinoma cell culture challenges the association of cancer stem cells with epithelial-to-mesenchymal transition

The cancer stem cell (CSC) and epithelial-to-mesenchymal transition (EMT) models have been closely associated and used to describe both the formation of metastasis and therapy resistance. We established a primary lung cell culture from a patient in a clinically rare and unique situation of primary resistant disease. This culture consisted of two biologically profoundly distinct adenocarcinoma cell subpopulations, which differed phenotypically and genotypically. One subpopulation initiated and sustained in spheroid cell culture (LT22s) whereas the other subpopulation was only capable of growth and proliferation under adherent conditions (LT22a). In contrast to our expectations, LT22s were strongly associated with the epithelial phenotype, and expressed additionally CSC markers ALDH1 and CD133, whereas the LT22a was characterized as mesenchymal with lack of CSC markers. The LT22s cells also demonstrated an invasive behavior and mimicked gland formation. Finally, LT22s were more resistant to Cisplatin than LT22a cells. We demonstrate a primary lung adenocarcinoma cell culture derived from a patient with resistant disease, with epithelial aggressive subpopulation of cells associated with stem cell features and therapy resistance. Our findings challenge the current model associating CSC and disease resistance mainly to mesenchymal cells and may have important clinical implications.

PD-1 and PD-L1 Protein Expression Predict Survival in Completely Resected Lung Adenocarcinoma

&NA; The prognostic value of programmed cell death 1 (PD‐1) and/or programmed cell death ligand 1 (PD‐L1) expression in completely resected lung adenocarcinoma is still unclear. The expression of PD‐1 and PD‐L1 was determined using immunohistochemistry in 161 patients with lung adenocarcinoma. PD‐1 and PD‐L1 expression was associated with favorable overall survival. This knowledge could be important in the design of future clinical trials evaluating immune checkpoint inhibitors. Background: We assessed the prognostic value of programmed cell death 1 (PD‐1) and programmed cell death ligand 1 (PD‐L1) in patients with completely resected lung adenocarcinoma. Patients and Methods: PD‐1 and PD‐L1 expression was determined using immunohistochemistry in formalin‐fixed paraffin‐embedded surgical specimens and correlated with the clinicopathologic features and survival of 161 patients with lung adenocarcinoma. Results: PD‐1 expression on immune cells was observed in 71 of 159 evaluable tumor samples (45%) and was not significantly associated with the clinicopathologic features. Multivariate analyses identified PD‐1 expression as an independent prognostic factor for recurrence (adjusted hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.36‐0.95; P = .03) and death (adjusted HR, 0.48; 95% CI, 0.27‐0.86; P = 0.01). PD‐L1 expression on tumor cells was seen in 59 of 161 cases (37%) and correlated with KRAS mutation status (P = .02) and type of surgery (P = .01). PD‐L1 expression was not associated with recurrence‐free survival in the patients (adjusted HR, 0.90; 95% CI, 0.55‐1.48; P = .68) but correlated with longer overall survival (adjusted HR, 0.54; 95% CI, 0.30‐0.97; P = .04). Conclusion: PD‐1 and PD‐L1 expression was associated with favorable overall survival in patients with completely resected adenocarcinoma of the lung.

Frequency and clinical impact of preoperative circulating tumor cells in resectable non-metastatic lung adenocarcinomas

OBJECTIVES Despite successful surgery, 30-50% of patients with resectable non-small cell lung cancer develop tumor recurrence within 5 years of surgery. MATERIALS AND METHODS In this prospective study, we performed CTC enumerations in 40 patients with non-metastatic lung adenocarcinoma (NMLA) using a size-based microfilter. Additionally, cfDNA isolated from plasma was analyzed in 35 out of 40 patients. RESULTS CTCs were identified in 15 out of 40 patients (37.5%) with a range of 1-44 cells, whereas mutated cfDNA was only detected in 3 out of 35 patients (8.6%). Disease-free survival (DFS) was significantly associated with CTC positivity (log-rank p=0.025), grading (log-rank p=0.019), tumor stage (log-rank p=0.025) and lymph node status (log-rank p=0.029). Multivariate analysis, including tumor stage and grading, showed that CTC positivity (p=0.006), grading (0.039) and tumor stage (p=0.022) were independently associated with DFS. CONCLUSION Our study found that microfilter-based CTC enumeration in NMLA patients is an independent predictor of worse DFS. The used NGS-based cfDNA characterization had limited sensitivity to be clinically informative in our study cohort. CTC assessment before surgery can thus identify NMLA patients at high risk of disease recurrence.

Non-sebaceous lymphadenoma of the lacrimal gland: first report of a new localization

Tumors of the lacrimal gland are rare, with an incidence of less than 1 per 1,000,000 individuals per year [1]. They represent 6–12% of all orbital space-occupying lesions. Approximately 22–28% of these are primary epithelial tumors [2–4]. Since lacrimal gland tumors generally recapitulate the clinicopathologic features of their salivary gland counterparts, the World Health Organization (WHO) classification of salivary gland tumors can be applied to these tumors as well [5]. Fifty percent of primary epithelial tumors of the lacrimal gland are malignant. The most frequently encountered type is adenoid cystic carcinoma, which comprises approximately 20–30% of malignant neoplasms [6]. However, a variety of malignant tumor types that mirror their salivary gland counterparts have been described including ductal carcinoma, acinic cell carcinoma, primary squamous cell carcinoma, mucoepidermoid carcinoma, oncocytic carcinoma, polymorphous low-grade adenocarcinoma, carcinoma ex pleomorphic adenoma, myoepithelial carcinoma, lymphoepithelial carcinoma, epithelial-myoepithelial carcinoma, cystadenocarcinoma, primary sebaceous adenocarcinoma, and basal cell adenocarcinoma. On the other hand, the most common benign tumor is pleomorphic adenoma, which comprises around 50% of all epithelial tumors [3, 4]. Other benign salivary type tumors that have been described in lacrimal gland are exceptionally rare [7] and include oncocytoma, cystadenoma, myoepithelioma, and Warthin tumor, also known as papillary cystadenoma lymphomatosum. Here, we present, to our knowledge, the first description of a case of non-sebaceous lymphadenoma of the lacrimal gland.

Immune cell landscape in therapy-naïve squamous cell and adenocarcinomas of the lung

Squamous cell and adenocarcinomas of the lung develop different mechanisms during carcinogenesis to evade attacks of the immune system. Besides the well-known check-point control programmed death 1 and its ligand, many more mechanisms, acting either tumoricidal or in favor of tumor progression, exist. Analysis of the immune cell profiles in resected tissues and bronchoalveolar lavage samples and correlation between them and with overall survival data was performed. In all tumor samples in this study, cells of the immune system expressed a tumor-cooperating phenotype. High numbers of regulatory T cells, or alternatively expression of Vista on lymphocytes was present. Tumoricidal dendritic cells were absent in tumor tissue, and barely present in bronchoalveolar lavage, whereas tumor-friendly monocytoid and plasmocytoid dendritic cells were seen in both. Alveolar macrophages were predominantly differentiated into tumor-cooperating M2 types, whereas tumoricidal M1 macrophages were absent or rare. The expression of PDL1 on tumor cells did not correlate with any other immune cells. Expression of PD1 on lymphocytes was frequently encountered. None of analyzed immune cells showed correlation with overall survival. Immune cells in bronchoalveolar lavage and tissue did not correlate. For the first time, a tissue-based analysis of different immune cells in squamous cell and adenocarcinomas of the lung is provided, trying to explain their potential role in tumor development and progression. Discordant numbers of cells with bronchoalveolar lavage are most probably due to the fact that bronchoalveolar lavage reflects the situation in the whole lung, where chronic obstructive lung disease and other conditions are present.

Afatinib restrains K-RAS–driven lung tumorigenesis

K-RAS–mutated lung adenocarcinomas depend on ERBB signaling, and pan-ERBB inhibitors impair K-RAS–driven lung tumorigenesis. A new role for kinase inhibitors The K-RAS oncogene is frequently mutated in a variety of cancer types, including lung cancer. Lung cancers with K-RAS mutations are usually difficult to target, and conventional thinking dictates that these tumors are resistant to receptor tyrosine kinase inhibitors because those act upstream of the constitutively active K-RAS protein. However, it appears that receptor tyrosine kinase signaling may have an effect on K-RAS–driven lung tumors after all, by amplifying their growth beyond the effects of K-RAS alone. Kruspig et al. and Moll et al. independently reached this conclusion and identified approved multikinase inhibitors that are effective in the setting of K-RAS–mutant lung cancer in multiple mouse models, suggesting that this may be a potential treatment strategy for human patients as well. On the basis of clinical trials using first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), it became a doctrine that V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) mutations drive resistance to EGFR inhibition in non–small cell lung cancer (NSCLC). Conversely, we provide evidence that EGFR signaling is engaged in K-RAS–driven lung tumorigenesis in humans and in mice. Specifically, genetic mouse models revealed that deletion of Egfr quenches mutant K-RAS activity and transiently reduces tumor growth. However, EGFR inhibition initiates a rapid resistance mechanism involving non-EGFR ERBB family members. This tumor escape mechanism clarifies the disappointing outcome of first-generation TKIs and suggests high therapeutic potential of pan-ERBB inhibitors. On the basis of various experimental models including genetically engineered mouse models, patient-derived and cell line–derived xenografts, and in vitro experiments, we demonstrate that the U.S. Food and Drug Administration–approved pan-ERBB inhibitor afatinib effectively impairs K-RAS–driven lung tumorigenesis. Our data support reconsidering the use of pan-ERBB inhibition in clinical trials to treat K-RAS–mutated NSCLC.

Candida infection of membrane oxygenator during ECMO therapy

First study able to assign Candidemia to the membrane oxygenator of the ECMO device Proof of infection by pre- and post oxygenator blood cultures and histologic analysis This infection represents a subentity of ECMO Device Related Bloodstream Infections.

Influence of eukaryotic translation initiation factor 6 on non–small cell lung cancer development and progression

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Dysregulation of protein synthesis plays a major role in carcinogenesis, a process regulated at multiple levels, including translation of mRNA into proteins. Ribosome assembly requires correct association of ribosome subunits, which is ensured by eukaryotic translation initiation factors (eIFs). eIFs have become targets in cancer therapy studies, and promising data on eIF6 in various cancer entities have been reported. Therefore, we hypothesised that eIF6 represents a crossroad for pulmonary carcinogenesis. High levels of eIF6 are associated with shorter patient overall survival in adenocarcinoma (ADC), but not in squamous cell carcinoma (SQC) of the lung. We demonstrate significantly higher protein expression of eIF6 in ADC and SQC than in healthy lung tissue based on immunohistochemical data from tissue microarrays (TMAs) and on fresh frozen lung tissue. Depletion of eIF6 in ADC and SQC lung cancer cell lines inhibited cell proliferation and induced apoptosis. Knockdown of eIF6 led to pre-rRNA processing and ribosomal 60S maturation defects. Our data indicate that eIF6 is upregulated in NSCLC, suggesting an important contribution of eIF6 to the development and progression of NSCLC and a potential for new treatment strategies against NSCLC.

FGF2 and EGF induce epithelial–mesenchymal transition in malignant pleural mesothelioma cells via a MAPKinase/MMP1 signal

Malignant pleural mesothelioma (MPM), an aggressive malignancy affecting pleural surfaces, occurs in three main histological subtypes. The epithelioid and sarcomatoid subtypes are characterized by cuboid and fibroblastoid cells, respectively. The biphasic subtype contains a mixture of both. The sarcomatoid subtype expresses markers of epithelial-mesenchymal transition (EMT) and confers the worst prognosis, but the signals and pathways controlling EMT in MPM are not well understood. We demonstrate that treatment with FGF2 or EGF induced a fibroblastoid morphology in several cell lines from biphasic MPM, accompanied by scattering, decreased cell adhesion and increased invasiveness. This depended on the MAP-kinase pathway but was independent of TGFβ or PI3-kinase signaling. In addition to changes in known EMT markers, microarray analysis demonstrated differential expression of MMP1, ESM1, ETV4, PDL1 and BDKR2B in response to both growth factors and in epithelioid versus sarcomatoid MPM. Inhibition of MMP1 prevented FGF2-induced scattering and invasiveness. Moreover, in MPM cells with sarcomatoid morphology, inhibition of FGF/MAP-kinase signaling induced a more epithelioid morphology and gene expression pattern. Our findings suggest a critical role of the MAP-kinase axis in the morphological and behavioral plasticity of mesothelioma.