Gene Expression-Based Approaches in Differentiation of Metastases and Second Primary Tumour

A 64-year-old male patient was diagnosed with 3 consecutive non-small cell lung carcinomas (NSCLC). In the current study, we applied whole-genome gene expression analysis to control, primary and locally recurrent cancer, and supposed metastasis samples of a single patient. According to our knowledge, there are no published papers describing the gene expression profiles of a single patient's squamous cell lung cancers. As the histology and differentiation grade of the primary cancer and the supposed metastasis differed minimally, but local recurrence was poorly differentiated, molecular profiling of the samples was carried out in order to confirm or reject the hypothesis of second primary cancer. Principal component analysis of the gene expression data revealed distinction of the local recurrence. Gene ontology analysis showed no molecular characteristics of metastasis in the supposed metastasis. Gene expression analysis is valuable and can be supportive in decision-making of diagnostically complicated cancer cases

Methylation Markers of Early-Stage Non-Small Cell Lung Cancer

Despite of intense research in early cancer detection, there is a lack of biomarkers for the reliable detection of malignant tumors, including non-small cell lung cancer (NSCLC). DNA methylation changes are common and relatively stable in various types of cancers, and may be used as diagnostic or prognostic biomarkers.We performed DNA methylation profiling of samples from 48 patients with stage I NSCLC and 18 matching cancer-free lung samples using microarrays that cover the promoter regions of more than 14,500 genes. We correlated DNA methylation changes with gene expression levels and performed survival analysis.We observed hypermethylation of 496 CpGs in 379 genes and hypomethylation of 373 CpGs in 335 genes in NSCLC. Compared to adenocarcinoma samples, squamous cell carcinoma samples had 263 CpGs in 223 hypermethylated genes and 513 CpGs in 436 hypomethylated genes. 378 of 869 (43.5%) CpG sites discriminating the NSCLC and control samples showed an inverse correlation between CpG site methylation and gene expression levels. As a result of a survival analysis, we found 10 CpGs in 10 genes, in which the methylation level differs in different survival groups.We have identified a set of genes with altered methylation in NSCLC and found that a minority of them showed an inverse correlation with gene expression levels. We also found a set of genes that associated with the survival of the patients. These newly-identified marker candidates for the molecular screening of NSCLC will need further analysis in order to determine their clinical utility

Influence of common genetic variation on lung cancer risk: meta-analysis of 14 900 cases and 29 485 controls

Recent genome-wide association studies (GWASs) have identified common genetic variants at 5p15.33, 6p21-6p22 and 15q25.1 associated with lung cancer risk. Several other genetic regions including variants of CHEK2 (22q12), TP53BP1 (15q15) and RAD52 (12p13) have been demonstrated to influence lung cancer risk in candidate- or pathway-based analyses. To identify novel risk variants for lung cancer, we performed a meta-analysis of 16 GWASs, totaling 14 900 cases and 29 485 controls of European descent. Our data provided increased support for previously identified risk loci at 5p15 (P = 7.2 × 10−16), 6p21 (P = 2.3 × 10−14) and 15q25 (P = 2.2 × 10−63). Furthermore, we demonstrated histology-specific effects for 5p15, 6p21 and 12p13 loci but not for the 15q25 region. Subgroup analysis also identified a novel disease locus for squamous cell carcinoma at 9p21 (CDKN2A/p16INK4A/p14ARF/CDKN2B/p15INK4B/ANRIL; rs1333040, P = 3.0 × 10−7) which was replicated in a series of 5415 Han Chinese (P = 0.03; combined analysis, P = 2.3 × 10−8). This large analysis provides additional evidence for the role of inherited genetic susceptibility to lung cancer and insight into biological differences in the development of the different histological types of lung cance

Mitteväikerakulise kopsuvähi kogugenoomi geeniekspressiooni- ja assotsiatsiooniuuringud

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Maailmas avastatakse igal aastal 1.6 miljonit uut kopsuvähi juhtu, Eestis ligikaudu 700.Kuigi kopsuvähi peamisteks riskifaktoriteks on suitsetamine ja õhusaaste, kokkupuude asbesti ja raskmetallidega, mängib haiguse tekkes ja arengus olulist rolli ka indiviidi geneetiline taust. Kopsuvähi diagnoosi ja prognoosi aluseks on TNM klassifikatsioon ja histoloogiline analüüs, kuid gruppide sisene ravivastus ja elulemus väga varieeruvad. Samuti esineb märkimisväärsel hulgal vähkkasvajaid, mille histoloogiline määratlemine on võimatu, kas siis dediferentseerumise või segatüübilise kasvaja tõttu. Sellest tulenevalt on asutud otsima uusi prognostilisi ja diagnostilisi molekulaarseid markereid nii DNA kui ka RNA näol, mis võimaldaksid täpsemalt klassifitseerida kopsuvähki ja juhtida raviarsti sobilikuma ravi valikul. Käesolevas doktoritöös on kasutatud kogu genoomi hõlmavat assotsiatsiooni- ja geeniekspressiooni analüüsi eesmärkidega identifitseerida kopsuvähi riskialleele ning vähkkasvajate spetsiifilisi geeniekspressiooni profiile. Käesolevas töös käsitletud assostsiatsiooniuuringu metaanalüüsi käigus kasutati 13 300 kopsuvähi ja 19 666 kontrollindiviidi genoomi andmeid, mille tulemusel leidsid kinnitust kõik eelnevalt identifitseeritud kopsuvähi riskilookused. Samuti tuvastati uusi potentsiaalseid kopsuvähiga seotud kromosoomilookuseid. Käesoleva töö raames teostatud kogugenoomi geeniekspressiooni analüüsil identifitseeriti uued potentsiaalsed kopsuvähi biomarkerid ning molekulaarsed profiilid, mis ennustasid patsientide elulemust paremini kui histoloogiline klassifitseerimine. Lisaks avastati, et patsiendid, kellede adenokartsinoomi proovides oli RNA lagunenud, omasid ka statistiliselt olulist kehvemat prognoosi. Käesoleva doktoritöö kolmandas artiklis on uuritud ühe patsiendi kahe kontrollkoe ja kolme kopsuvähi kasvaja kogugenoomi geeniekspressiooni profiile selgitamaks metastaasi või teise primaarkasvaja hüpoteesi. Lisaks geeniekspressiooni profiilide rakendamise testimisest personaalses meditsiinis oli töö ajendatud ka asjaolust, et nii prognoos kui ka ravistrateegia on metastaseerunud ja mittemetastaseerunud kopsuvähi korral väga erinev. Geeniontoloogia, peakomponent- ning korrelatsioonianalüüs viitasid üheselt uue primaarkasvaja tekkele, mida kinnitab ka tänaseni säilinud patsiendi suhteliselt hea kliiniline pilt. Maailmas avastatakse igal aastal 1.6 miljonit uut kopsuvähi juhtu, Eestis ligikaudu 700.Kuigi kopsuvähi peamisteks riskifaktoriteks on suitsetamine ja õhusaaste, kokkupuude asbesti ja raskmetallidega, mängib haiguse tekkes ja arengus olulist rolli ka indiviidi geneetiline taust. Kopsuvähi diagnoosi ja prognoosi aluseks on TNM klassifikatsioon ja histoloogiline analüüs, kuid gruppide sisene ravivastus ja elulemus väga varieeruvad. Samuti esineb märkimisväärsel hulgal vähkkasvajaid, mille histoloogiline määratlemine on võimatu, kas siis dediferentseerumise või segatüübilise kasvaja tõttu. Sellest tulenevalt on asutud otsima uusi prognostilisi ja diagnostilisi molekulaarseid markereid nii DNA kui ka RNA näol, mis võimaldaksid täpsemalt klassifitseerida kopsuvähki ja juhtida raviarsti sobilikuma ravi valikul. Käesolevas doktoritöös on kasutatud kogu genoomi hõlmavat assotsiatsiooni- ja geeniekspressiooni analüüsi eesmärkidega identifitseerida kopsuvähi riskialleele ning vähkkasvajate spetsiifilisi geeniekspressiooni profiile. Käesolevas töös käsitletud assostsiatsiooniuuringu metaanalüüsi käigus kasutati 13 300 kopsuvähi ja 19 666 kontrollindiviidi genoomi andmeid, mille tulemusel leidsid kinnitust kõik eelnevalt identifitseeritud kopsuvähi riskilookused. Samuti tuvastati uusi potentsiaalseid kopsuvähiga seotud kromosoomilookuseid. Käesoleva töö raames teostatud kogugenoomi geeniekspressiooni analüüsil identifitseeriti uued potentsiaalsed kopsuvähi biomarkerid ning molekulaarsed profiilid, mis ennustasid patsientide elulemust paremini kui histoloogiline klassifitseerimine. Lisaks avastati, et patsiendid, kellede adenokartsinoomi proovides oli RNA lagunenud, omasid ka statistiliselt olulist kehvemat prognoosi. Käesoleva doktoritöö kolmandas artiklis on uuritud ühe patsiendi kahe kontrollkoe ja kolme kopsuvähi kasvaja kogugenoomi geeniekspressiooni profiile selgitamaks metastaasi või teise primaarkasvaja hüpoteesi. Lisaks geeniekspressiooni profiilide rakendamise testimisest personaalses meditsiinis oli töö ajendatud ka asjaolust, et nii prognoos kui ka ravistrateegia on metastaseerunud ja mittemetastaseerunud kopsuvähi korral väga erinev. Geeniontoloogia, peakomponent- ning korrelatsioonianalüüs viitasid üheselt uue primaarkasvaja tekkele, mida kinnitab ka tänaseni säilinud patsiendi suhteliselt hea kliiniline pilt.There are more than 1.6 million and 700 new cases of lung cancer in the World and Estonia diagnosed every year respectively. Although, the main risk factors of lung cancer are smoking and air pollution, exposure to asbestos and heavy metals, the individuals genetic background plays also an important role. Currently applied basis for the diagnosis and prognosis of lung are TNM classification and histologycal evaluation but substantial number of patients within the specific group have different treatment responses and survival. Moreover, there are also large number of cancer samples that lack the histologycal and morphologycal features to be adequately classified. Therefore, the discovery of new DNA and RNA based diagnostic and prognostic markes are extensively sought to facilitate the accurate classification and personalised medicine in the field. Whole genome gene expression profiling of lung cancer samples and –association analysis of DNA markers of the patients was carried out in the current study. As a result of the association study all the previously identyfied lung cancer risk locuses were confirmed. In addition, several new potential lung cancer associated loci were highlighted. In the lung cancer whole genome gene expression analysis study the new potential biomarkers and molecular profiles were identified, that predicted the patients survival with better prognostic value than histologycal classification. In addition, for the patients with adenocarcinoma, the RNA degradation was found to have statistically significant poor prognosis. In the case study of whole genome gene expression, the patien´s two control and three cancer samples were analysed simultaneously, in aim to support or reject the hypothesis of second primary versus metastatsis, that is crucial in treatment selection. The gene expression data analysis methods like gene ontology, principal component analysis and correlation analysis were applied, that all indicated to the presence of new primary cancer. The result is also supported by the relatively good clinical performance of the patient. Therefore, whole genome gene expression analysis may be supportiv in assessing the clinically difficult cancer samples

Identification of MiR-374a as a prognostic marker for survival in patients with early-stage nonsmall cell lung cancer

Lung cancer is one of the deadliest types of cancer proven by the poor survival and high relapse rates after surgery. Recently discovered microRNAs (miRNAs), small noncoding RNA molecules, play a crucial role in modulating gene expression networks and are directly involved in the progression of a number of human cancers. In this study, we analyzed the expression profile of 858 miRNAs in 38 Estonian nonsmall cell lung cancer (NSCLC) samples (Stage I and II) and 27 adjacent nontumorous tissue samples using Illumina miRNA arrays. We found that 39 miRNAs were up-regulated and 33 down-regulated significantly in tumors compared with normal lung tissue. We observed aberrant expression of several well-characterized tumorigenesis-related miRNAs, as well as a number of miRNAs whose function is currently unknown. We show that low expression of miR-374a in early-stage NSCLC is associated with poor patient survival. The combinatorial effect of the up- and down-regulated miRNAs is predicted to most significantly affect pathways associated with cell migration, differentiation and growth, and several signaling pathways that contribute to tumorigenesis. In conclusion, our results demonstrate that expression of miR-374a at early stages of NSCLC progression can serve as a prognostic marker for patient risk stratification and may be a promising therapeutic target for the treatment of lung cancer

Correction: Methylation Markers of Early-Stage Non-Small Cell Lung Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0039813.]

Survival curves of 10 differentially methylated CpG sites.

<p>We performed a survival test on each of the CpG sites. The methylation values are divided into 3 groups: low (0–0.25), medium (0.25–0.75) and high (0.75–1). As a result we found 10 CpG sites whose methylation level differs in different survival groups. The x-axis shows survival in years and the y-axis shows overall survival.</p