SOX9 has distinct roles in the formation and progression of different non-small cell lung cancer histotypes.

The transcription factor SOX9 is a key regulator of multiple developmental processes and is frequently re-expressed in non-small cell lung cancer (NSCLC). Its precise role in the progression of NSCLC histotypes has, however, remained elusive. We show that SOX9 expression relates to poor overall survival and invasive histopathology in human non-mucinous adenocarcinoma and is absent in murine early minimally invasive and low in human in situ adenocarcinoma. Interestingly, despite wide SOX9 expression across advanced NSCLC histotypes, its genetic deletion in the murine KrasG12D ;Lkb1fl/fl model selectively disrupted only the growth of papillary NSCLC, without affecting the initiation of precursor lesions or growth of mucinous or squamous tissue. Spatial tissue phenotyping indicated a requirement of SOX9 expression for the progression of surfactant protein C-expressing progenitor cells, which gave rise to papillary tumours. Intriguingly, while SOX9 expression was dispensable for squamous tissue formation, its loss in fact led to enhanced squamous tumour metastasis, which was associated with altered collagen IV deposition in the basement membrane. Our work therefore demonstrates histopathology-selective roles for SOX9 in NSCLC progression, namely as a promoter for papillary adenocarcinoma progression, but an opposing metastasis-suppressing role in squamous histotype tissue. This attests to a pleiotropic SOX9 function, linked to the cell of origin and microenvironmental tissue contexts. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Performance of Finnish biobanks in nationwide pulmonary carcinoid tumour research.

Finnish hospital-integrated biobanks administer millions of formalin-fixed paraffin-embedded tissue samples collected within the clinical diagnostics. According to the Finnish Biobank Act, these samples can be coupled with patients' clinical follow-up data and the data retrieved from national health registries. We collected a nationwide pulmonary carcinoid tumour series from Finnish biobanks to study prognostic factors as well as to explore how the number of tumours found in the Finnish biobanks corresponds to the number of tumours registered by the Finnish Cancer Registry (FCR). Finnish biobanks identified 88% of the tumours registered by the FCR and were able to deliver 63%. The main reasons for lacking samples were paucity of resected primary tumour tissue, incompatible primary diagnosis, and the absence of tissue blocks in the archives. The main bottleneck in the sample application process was retrieving patient data. Altogether, we received 224 tumour samples with appropriate patient data and identified six prognostic factors for shorter disease-specific survival: age over 56 years at the time of diagnosis, tumour size over 2.5 cm, atypical histology, Ki-67 proliferation index higher than 2.5%, hilar/mediastinal lymph node involvement at the time of diagnosis, and the presence of metastatic disease. In conclusion, the Finnish biobank infrastructure offers excellent opportunities for tissue-based research. However, to be able to develop the biobank operations further, involving more medical knowledge in the sample and data acquisition process is a necessity. Also, when working with tissue samples collected over decades, histological expertise is essential for re-evaluation and re-classification of the samples.

Receptor Tyrosine Kinase Signaling Networks Define Sensitivity to ERBB Inhibition and Stratify Kras-Mutant Lung Cancers.

Most non-small cell lung cancers (NSCLC) contain nontargetable mutations, including KRAS, TP53, or STK11/LKB1 alterations. By coupling ex vivo drug sensitivity profiling with in vivo drug response studies, we aimed to identify drug vulnerabilities for these NSCLC subtypes. Primary adenosquamous carcinoma (ASC) or adenocarcinoma (AC) cultures were established from KrasG12D/+;Lkb1fl/fl (KL) tumors or AC cultures from KrasG12D/+;p53fl/fl (KP) tumors. Although p53-null cells readily propagated as conventional cultures, Lkb1-null cells required conditional reprograming for establishment. Drug response profiling revealed short-term response to MEK inhibition, yet long-term clonogenic assays demonstrated resistance, associated with sustained or adaptive activation of receptor tyrosine kinases (RTK): activation of ERBBs in KL cultures, or FGFR in AC cultures. Furthermore, pan-ERBB inhibition reduced the clonogenicity of KL cultures, which was exacerbated by combinatorial MEK inhibition, whereas combinatorial MEK and FGFR inhibition suppressed clonogenicity of AC cultures. Importantly, in vivo studies confirmed KL-selective sensitivity to pan-ERBB inhibition, which correlated with high ERBB ligand expression and activation of ERBB receptors, implying that ERBB network activity may serve as a predictive biomarker of drug response. Interestingly, in human NSCLCs, phosphorylation of EGFR or ERBB3 was frequently detected in ASCs and squamous cell carcinomas. We conclude that analysis of in situ ERBB signaling networks in conjunction with ex vivo drug response profiling and biochemical dissection of adaptive RTK activities may serve as a valid diagnostic approach to identify tumors sensitive to ERBB network inhibition.

PD-1 and PD-L1 expression in pulmonary carcinoid tumors and their association to tumor spread.

Pulmonary carcinoid (PC) tumors are rare tumors that account for approximately 1% of all lung cancers. The primary treatment option is surgery, while there is no standard treatment for metastatic disease. As the number of PCs diagnosed yearly is increasing, there is a need to establish novel therapeutic options. This study aimed to investigate programmed death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) expression in PC tumors since blocking of the PD-1/PD-L1 pathway is a promising therapeutic option in various other malignancies. A total of 168 PC patients treated between 1990 and 2013 were collected from the Finnish biobanks. After re-evaluation of the tumors, 131 (78%) were classified as typical carcinoid (TC) and 37 (22%) as atypical carcinoid (AC) tumors. Primary tumor samples were immunohistochemically labeled for PD-1, PD-L1 and CD8. High PD-1 expression was detected in 16% of the tumors. PD-L1 expression was detected in 7% of TC tumors; all AC tumors were PD-L1 negative. PD-L1 expression was associated with mediastinal lymph-node metastasis at the time of diagnosis (P = 0.021) as well as overall metastatic potential of the tumor (P = 0.010). Neither PD-1 expression, PD-L1 expression nor CD8+ T cell density was associated with survival. In conclusion, PD-1 and PD-L1 were expressed in a small proportion of PC tumors and PD-L1 expression was associated with metastatic disease. Targeting of the PD-1/PD-L1 pathway with immune checkpoint inhibitors may thus offer a treatment option for a subset of PC patients.

CDKN2A copy number and p16 expression in malignant pleural mesothelioma in relation to asbestos exposure.

BACKGROUND: Deletion of the CDKN2A locus is centrally involved in the development of several malignancies. In malignant pleural mesothelioma (MPM), it is one of the most frequently reported genomic alteration. MPM is strongly associated with a patients' asbestos exposure. However, the status of CDKN2A and the expression of the corresponding protein, p16, in relation to MPM patient's asbestos exposure is poorly known. Copy number alterations in 2p16, 9q33.1 and 19p13 have earlier been shown to accumulate in lung cancer in relation to asbestos exposure but their status in MPM is unclear. METHODS: We studied DNA copy numbers for CDKN2A using fluorescence in situ hybridization (FISH) and p16 expression by immunohistochemistry (IHC) in 92 MPM patients, 75 of which with known asbestos exposure status. We also studied, in MPM, copy number alterations in 2p16, 9q33.1 and 19p13 by FISH. RESULTS: We were unable to detect an association between p16 expression and pulmonary asbestos fiber count in MPM tumor cells. However, significantly more MPM patients with high pulmonary asbestos fiber count (> 1 million fibers per gram [f/g]) had stromal p16 immunoreactivity than MPM of patients with low exposure (≤ 0.5 million f/g) (51.4% vs 16.7%; p = 0.035, Chi-Square). We found that an abnormal copy number of CDKN2A in MPM tumor cells associated with a high pulmonary asbestos fiber count (p = 0.044, Fisher's Exact test, two-tailed). In contrast to our earlier findings in asbestos associated lung cancer, DNA copy number changes in 2p16, 9q33 and 19p13 were not frequent in MPM although single cases with variable copy numbers on those regions were seen. CONCLUSIONS: We found two instances where the gene locus CDKN2A or its corresponding protein expression, is associated with high asbestos exposure levels. This suggests that there may be biological differences between the mesotheliomas with high pulmonary asbestos fiber count and those with low fiber count.

Somatostatin Receptor Expression Is Associated With Metastasis and Patient Outcome in Pulmonary Carcinoid Tumors.

CONTEXT: Pulmonary carcinoids (PCs) belong to neuroendocrine tumors that often overexpress somatostatin receptors (SSTRs). This overexpression provides a molecular basis for tumor imaging and treatment with somatostatin analogs. OBJECTIVE: To evaluate SSTR1 to SSTR5 distribution in a large set of PC tumors and to investigate whether the expression is associated with clinicopathological and outcome data. DESIGN, SETTING, AND PATIENTS: This retrospective study was conducted at Helsinki University Hospital and University of Helsinki. It included 178 PC tumors coupled with patients' clinical data retrieved through Finnish biobanks. After histological reclassification, tissue specimens were processed into next-generation tissue microarray format and stained immunohistochemically with monoclonal SSTR1 to SSTR5 antibodies. MAIN OUTCOME MEASURE: SSTR1 to SSTR5 expression in PC tumors. RESULTS: Expression of SSTR1 to SSTR5 was detected in 52%, 75%, 56%, 16%, and 32% of the tumors, respectively. Membrane-bound staining was observed for all receptors. SSTR2 negativity and SSTR4 positivity was associated with lymph node involvement at the time of surgery (P = 0.014 and P = 0.017, respectively) and with distant metastasis (P = 0.027 and P = 0.015, respectively). SSTR3 and SSTR4 expression was associated with increased risk of shorter survival [P = 0.046, hazard ratio (HR) 4.703, 95% CI 1.027 to 21.533; and P = 0.013, HR 6.64, 95% CI 1.48 to 29.64, respectively], whereas expression of SSTR1 and SSTR2 was associated with improved outcome (P = 0.021, HR 0.167, 95% CI 0.037 to 0.765; and P = 0.022, HR 0.08, 95% CI 0.01 to 0.70, respectively). CONCLUSION: SSTR1 to SSTR5 expression is observed in PCs. As SSTR expression is associated with the tumor's metastatic potential and patient outcome, these receptors may offer the possibility for individualized prognosis estimation.

Spatial aspects of oncogenic signalling determine the response to combination therapy in slice explants from Kras-driven lung tumours.

A key question in precision medicine is how functional heterogeneity in solid tumours informs therapeutic sensitivity. We demonstrate that spatial characteristics of oncogenic signalling and therapy response can be modelled in precision-cut slices from Kras-driven non-small-cell lung cancer with varying histopathologies. Unexpectedly, profiling of in situ tumours demonstrated that signalling stratifies mostly according to histopathology, showing enhanced AKT and SRC activity in adenosquamous carcinoma, and mitogen-activated protein kinase (MAPK) activity in adenocarcinoma. In addition, high intertumour and intratumour variability was detected, particularly of MAPK and mammalian target of rapamycin (mTOR) complex 1 activity. Using short-term treatment of slice explants, we showed that cytotoxic responses to combination MAPK and phosphoinositide 3-kinase-mTOR inhibition correlate with the spatially defined activities of both pathways. Thus, whereas genetic drivers determine histopathology spectra, histopathology-associated and spatially variable signalling activities determine drug sensitivity. Our study is in support of spatial aspects of signalling heterogeneity being considered in clinical diagnostic settings, particularly to guide the selection of drug combinations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Clinicopathological indicators of survival among patients with pulmonary carcinoid tumor.

BACKGROUND: Pulmonary carcinoids (PC) are rare malignant neoplasms that cover approximately 1% of all lung cancers. PCs are classified by histological criteria as either typical (TC) or atypical (AC). Histological subtype is the most studied prognostic factor. The aim of this study was to evaluate if other tissue or clinical features are associated with patient outcomes. MATERIAL AND METHODS: We retrospectively reviewed clinical records of 133 PC patients who underwent operation in the Helsinki University Hospital between 1990 and 2013. Tissue specimens were re-evaluated, processed into tissue microarray format and stained immunohistochemically with serotonin, calcitonin, adrenocorticotropic hormone (ACTH), thyroid transcription factor-1 (TTF-1) and Ki-67. Survival and risk analyses were performed. RESULTS: Based on histology, 75% (n = 100) of the tumors were TCs and 25% (n = 33) ACs. TCs had higher 10-year disease-specific survival (DSS) rate than ACs (99% (95% CI, 93-100%) for TCs vs. 82% (95% CI, 61-92%) for ACs). Hormonally active tumors expressing serotonin, calcitonin or ACTH were noted in 53% of the specimens but hormonal expression was not associated with DSS. TTF-1 was positive in 78% of the specimens but was not associated with DSS. Ki-67 index varied between <1% and 15%. Ki-67 ≥ 2.5% was associated with shorter DSS (p = .004). The presence of metastatic disease (p = .001), tumor size ≥30 mm (p = .021) and atypical histology (p = .011) were also associated with disease-specific mortality. CONCLUSIONS: We conclude that PCs are uncommon tumors. When resected, the long-term survival is in general favorable. In this consecutive, single-institution cohort of patients, presence of metastatic disease, tumor size, histological subtype and Ki-67 index were associated with shorter disease-specific survival. As TC and AC have different clinical behaviors, the correct tumor classification at the time of diagnosis is a necessity.

Analysis of the Histologic Features Associated With Interobserver Variation in Idiopathic Pulmonary Fibrosis.

The histologic manifestation of idiopathic pulmonary fibrosis (IPF) is usual interstitial pneumonia (UIP), which is a good prognostic determinant of survival compared with other histologic interstitial lung disease patterns. According to the current international guidelines, the histologic features of suspected IPF/UIP are divided into 4 categories: UIP, probable UIP, possible UIP, and not UIP pattern. Four pulmonary pathologists who were blinded to clinicoradiologic information reevaluated 50 surgical lung biopsies (83.3%), 6 lung explant (10.0%), and 4 autopsy samples (6.7%) from the FinnishIPF registry (N=60) using the current diagnostic guidelines. Additional histologic features atypical for UIP were also evaluated and compared with clinicora-diologic information. The interobserver agreement of pathologists was examined by Cohen kappa (κ) coefficient; the survival of the patients was estimated with Kaplan-Meier curves. The histologic reevaluation indicated that 38 of 60 patients (63.3%) had definite UIP. Inflammation was the most common additional histologic finding (15/60, 25.0%). The interobserver agreement on histologic diagnosis ranged from slight (κ=0.044) to substantial (κ=0.779). The interobserver agreement varied extensively with regard to the presence of giant cells. The observed histologic features displayed no association with radiologic patterns or survival. Definite UIP and honeycombing findings in high-resolution computed tomography correlated with poor prognosis. A high level of interobserver variability was observed between pathologists, even in this well-defined cohort of IPF patients, which highlights the importance of multidisciplinary decision making in IPF diagnostics and stresses the need for a reassessment of the histologic criteria.

Asbestos-associated genome-wide DNA methylation changes in lung cancer.

Previous studies have revealed a robust association between exposure to asbestos and human lung cancer. Accumulating evidence has highlighted the role of epigenome deregulation in the mechanism of carcinogen-induced malignancies. We examined the impact of asbestos on DNA methylation. Our genome-wide studies (using Illumina HumanMethylation450K BeadChip) of lung cancer tissue and paired normal lung from 28 asbestos-exposed or non-exposed patients, mostly smokers, revealed distinctive DNA methylation changes. We identified a number of differentially methylated regions (DMR) and differentially variable, differentially methylated CpGs (DVMC), with individual CpGs further validated by pyrosequencing in an independent series of 91 non-small cell lung cancer and paired normal lung. We discovered and validated BEND4, ZSCAN31 and GPR135 as significantly hypermethylated in lung cancer. DMRs in genes such as RARB (FDR 1.1 × 10-19 , mean change in beta [Δ] -0.09), GPR135 (FDR 1.87 × 10-8 , mean Δ -0.09) and TPO (FDR 8.58 × 10-5 , mean Δ -0.11), and DVMCs in NPTN, NRG2, GLT25D2 and TRPC3 (all with p <0.05, t-test) were significantly associated with asbestos exposure status in exposed versus non-exposed lung tumors. Hypomethylation was characteristic to DVMCs in lung cancer tissue from asbestos-exposed subjects. When DVMCs related to asbestos or smoking were analyzed, 96% of the elements were unique to either of the exposures, consistent with the concept that the methylation changes in tumors may be specific for risk factors. In conclusion, we identified novel DNA methylation changes associated with lung tumors and asbestos exposure, suggesting that changes may be present in causal pathway from asbestos exposure to lung cancer.

Cell of Origin Links Histotype Spectrum to Immune Microenvironment Diversity in Non-small-Cell Lung Cancer Driven by Mutant Kras and Loss of Lkb1.

Lung cancers exhibit pronounced functional heterogeneity, confounding precision medicine. We studied how the cell of origin contributes to phenotypic heterogeneity following conditional expression of KrasG12D and loss of Lkb1 (Kras;Lkb1). Using progenitor cell-type-restricted adenoviral Cre to target cells expressing surfactant protein C (SPC) or club cell antigen 10 (CC10), we show that Ad5-CC10-Cre-infected mice exhibit a shorter latency compared with Ad5-SPC-Cre cohorts. We further demonstrate that CC10+ cells are the predominant progenitors of adenosquamous carcinoma (ASC) tumors and give rise to a wider spectrum of histotypes that includes mucinous and acinar adenocarcinomas. Transcriptome analysis shows ASC histotype-specific upregulation of pro-inflammatory and immunomodulatory genes. This is accompanied by an ASC-specific immunosuppressive environment, consisting of downregulated MHC genes, recruitment of CD11b+ Gr-1+ tumor-associated neutrophils (TANs), and decreased T cell numbers. We conclude that progenitor cell-specific etiology influences the Kras;Lkb1-driven tumor histopathology spectrum and histotype-specific immune microenvironment.

Incidence, Risk Factors, and Outcome of Life-Threatening Ventricular Arrhythmias in Giant Cell Myocarditis.

BACKGROUND: Ventricular tachyarrhythmias are characteristic of giant cell myocarditis, but their true incidence, predictors, and outcome are unknown. METHODS AND RESULTS: Our work involved 51 patients with giant cell myocarditis (35 women) aged 52±12 years. Their medical records were reviewed for history, results of laboratory and imaging studies, and occurrence of serious cardiac events, including life-threatening ventricular tachyarrhythmias. Sudden cardiac death (fatal or aborted) was the primary end point of our analyses, whereas the composite of sudden cardiac death and ventricular tachycardia requiring treatment constituted the secondary end point. Giant cell myocarditis presented as nonfatal ventricular tachyarrhythmia in 10 patients and as a fatal cardiac arrest in 1 patient. Overall, 14 of 50 patients suffered a sudden cardiac death during follow-up, with a cumulative incidence of 22% at 1 year and 26% at 5 years from presentation. The composite incidence of sudden cardiac death or ventricular tachycardia was 41% at 1 year and 55% at 5 years. The incidence of arrhythmias was associated with high plasma concentrations of troponin-T and N-terminal brain natriuretic propeptide, as well as with moderate-to-severe fibrosis on myocardial biopsy and history of ventricular tachyarrhythmias at presentation (P<0.05 for all). An intracardiac cardioverter defibrillator was implanted in 31 patients, of whom 17 had altogether 114 appropriate antiarrhythmic therapies by the device and none suffered an arrhythmic death. CONCLUSIONS: In giant cell myocarditis, the risk of life-threatening ventricular arrhythmias exceeds 50% at 5 years from admission, being related to the presenting clinical manifestation and markers of myocardial injury and scarring.

HuR in pheochromocytomas and paragangliomas - overexpression in verified malignant tumors.

Pheochromocytomas and paragangliomas are rare, neural crest-originating, neuroendocrine tumors. HuR is an mRNA-binding protein of the ELAV/Hu-protein family, which participates in posttranscriptional regulation of many cancer-associated genes. HuR expression has been connected with aggressive behavior of several malignancies. Cyclooxygenase-2 (COX-2) is also expressed in several malignant tumors, and its expression is regulated by HuR. Tissue microarray of 153 primary pheochromocytomas and paragangliomas was investigated for the expression of HuR and COX-2 proteins by immunohistochemistry using two different HuR antibodies (HuR19F12 and HuR3A). In these tumors, the expression of both intranuclear and cytoplasmic HuR was detectable. Increased cytoplasmic HuR expression was significantly associated with metastatic tumors. Increased COX-2 and MIB-1 expression also was associated with metastatic potential, and moreover, HuR and COX-2 expression correlated with each other. Our data suggest that increased expression of HuR protein is associated with metastatic potential of paragangliomas and pheochromocytomas, and COX-2 seems to be a target of HuR.

Long-term outcome and its predictors in giant cell myocarditis.

AIMS: There are no studies focusing on prognostic factors in giant cell myocarditis (GCM). We aimed to identify predictors of transplant-free survival in GCM. METHODS AND RESULTS: We analysed the details of 46 patients with GCM (31 women, mean age 51 ± 12 years) seen at our hospital since 1991 and followed for the occurrence of cardiac death or transplantation till May 2015. The association of transplant-free survival with patient characteristics, laboratory data on admission, and myocardial histology in the 38 patients diagnosed prior to death or transplantation was examined. Altogether 26 patients died (n = 8) or underwent transplantation (n = 18) a median of 11 months following symptom onset. The 5-year estimate of transplant-free survival was 42% [95% confidence interval (CI) 35-48%]. By Cox regression analysis, the hazard ratio for death or transplantation was 0.87 (95% CI 0.75-0.99) per +5% difference in LVEF, 1.06 (95% CI 1.03-1.10) per + 1000 ng/L difference in NT-proBNP, and 4.57 (95% CI 1.63-11.28) for cardiac troponin-T above the median of 85 ng/L at presentation. The severity of necrosis and fibrosis in myocardial biopsy, graded by the consensus of two cardiac pathologists as none, mild, moderate, or severe, predicted the outcome with a hazard ratio of 7.17 (95% CI 2.29-22.40) for the presence of either necrosis or fibrosis of at least moderate extent. CONCLUSIONS: In GCM, the probability of transplant-free survival is 42% at 5 years from symptom onset. Markers of myocyte injury and cardiac dysfunction help predict the outcome.

[Molecular pathologic diagnosis of lung cancer requires basic knowledge also from clinicians]. / Keuhkosyövän molekyylipatologinen diagnostiikka edellyttää perustietoja myös kliinikoilta.

Besides histological classification of lung cancers, molecular biological examination of tumors is part of modern diagnostics of lung cancers, and cancer therapies are based on molecular biological diagnosis. Current laboratory technique enables the examination of many mutations as part of standard routine diagnosis, necessitating functional multidisciplinary collaboration, with the pathologist playing a central role in the handling of the specimens. In the future, especially pathologists, pulmonary specialists and oncologists are expected to need a good command of molecular biological data on lung cancer, but basic knowledge will be required also from other physicians working with lung cancer patients.

[Diagnostics of non-small cell lung carcinoma]. / Ei-pienisoluisen keuhkosyöväin ALK-diagnostiikka.

ALK inhibitor therapy is individual cancer treatment, in which the targeted drug therapy is directed to a patient group that is likely to benefit from the therapy. The detection in the tumor of ALK gene (anaplastic lymphoma kinase) rearrangement is a prerequisite for the ALK inhibitor therapy for non-small cell lung carcinoma. The ALK assay should be performed for non-squamous cellular non-small cell lung carcinomas, especially adenocarcinomas. It is not recommended to be based on the patients' clinical features. The immunohistochemical method is well suited for screening of ALK positivity. The present recommendation is to ascertain the ALK gene rearrangement from immunopositive specimens by using the FISH procedure.

Targeted resequencing reveals ALK fusions in non-small cell lung carcinomas detected by FISH, immunohistochemistry, and real-time RT-PCR: a comparison of four methods.

Anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements occur in a subgroup of non-small cell lung carcinomas (NSCLCs). The identification of these rearrangements is important for guiding treatment decisions. The aim of our study was to screen ALK gene fusions in NSCLCs and to compare the results detected by targeted resequencing with results detected by commonly used methods, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and real-time reverse transcription-PCR (RT-PCR). Furthermore, we aimed to ascertain the potential of targeted resequencing in detection of ALK-rearranged lung carcinomas. We assessed ALK fusion status for 95 formalin-fixed paraffin-embedded tumor tissue specimens from 87 patients with NSCLC by FISH and real-time RT-PCR, for 57 specimens from 56 patients by targeted resequencing, and for 14 specimens from 14 patients by IHC. All methods were performed successfully on formalin-fixed paraffin-embedded tumor tissue material. We detected ALK fusion in 5.7% (5 out of 87) of patients examined. The results obtained from resequencing correlated significantly with those from FISH, real-time RT-PCR, and IHC. Targeted resequencing proved to be a promising method for ALK gene fusion detection in NSCLC. Means to reduce the material and turnaround time required for analysis are, however, needed.

Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS.

The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

Carbonic anhydrase IX in malignant pleural mesotheliomas: a potential target for anti-cancer therapy.

Malignant mesothelioma is a neoplasm deriving from mesothelial cells, which line the body cavities. The most common type is malignant pleural mesothelioma (MPM), which is a locally aggressive malignancy with poor prognosis. To improve both the clinical diagnostics and treatment it is necessary to identify novel molecular targets which are characteristic for MPM. Although carbonic anhydrase (CA) enzymes have been linked to pH regulation and spread of cancer cells, they have not been thoroughly studied in MPM specimens. We investigated by immunohistochemistry the expression of CA isozymes II, VII, IX, and XII in a series of 27 histological MPM tumor samples. CA IX was absent in the normal lung alveolar cells, whereas it was abundantly expressed in the normal pleural mesothelium and malignant mesothelioma cells. CA VII also showed weak or moderate reactions in several cases of mesotheliomas. Neither high expression of CA VII nor CA IX did correlate significantly with the survival of the patients. The very high expression of CA IX in MPM suggests that it could represent a novel molecular target for cancer research applications.

Accumulation of genomic alterations in 2p16, 9q33.1 and 19p13 in lung tumours of asbestos-exposed patients.

We have previously demonstrated an association between genomic alterations in 19p13, 2p16, and 9q33.1 and asbestos exposure in patients' lung tumours. This study detected allelic imbalance (AI) in these regions in asbestos-exposed lung cancer (LC) patients' histologically normal pulmonary epithelium. We extended the analyses of tumour tissue to cover a large LC patient cohort and studied DNA copy number alteration (CNA) and AI in 19p13, 2p16, and 9q33.1 for the first time in combination. We found both CNA and AI in ≥2/3 of the regions to be significantly and dose-dependently (P < 0.001) associated with pulmonary asbestos fibre count. Twenty percent of the exposed patients' LC showed CNA in ≥2/3 of the regions, whereas none of the non-exposed patients' LC showed CNA in more than one region. AI was evident in 89% of the exposed and in only 26% of the non-exposed patients' LC. The genomic alterations in 19p13, 2p16, and 9q33.1 in compilation identified asbestos-exposed patients' lung tumours better than each of the regions alone. These alterations form the basis for the development of a combinatorial molecular assay that could be used to identify asbestos-related LC.