Prognostic value of microRNA expression in operable non-small cell lung cancer patients.

BACKGROUND: About 50% of non-small cell lung cancer (NSCLC) patients develop distant metastases following pulmonary resection. Currently, there are no reliable factors allowing for individual selection of high-risk patients for adjuvant systemic therapies. METHODS: We assessed by quantitative reverse transcription PCR microRNA (miRNA) expression in 273 stage I-IIIA NSCLC samples. Expression of 677 miRNAs was evaluated in fresh-frozen tumour samples in the training cohort of 50 squamous cell carcinoma (SCC) patients who underwent curative surgery. Of those, 20 patients developed distant metastases, and 30 were free of recurrence for >4 years. In the second step, miRNAs with highest predictive value for distant relapse were re-evaluated in formalin-fixed paraffin-embedded material in an independent group of 134 stage I-IIIA SCC patients. Additionally, the same miRNAs were investigated in 89 lung adenocarcinoma (AC) patients and in normal lung parenchyma (NLP). RESULTS: In the training cohort of SCC, six miRNAs were differently expressed in the non-recurrent vs recurrent groups and correlated with distant recurrence-free survival, however none reached the level of significance after correction for multiple testing. Of these six miRNAs, miR-662, -192 and -192* were confirmed as prognostic in the independent SCC cohort. Expression of miR-128, -10b, -502-3p and -192 differed between SCC and AC, and miR-128 and -192 - between NLP and NSCLC. CONCLUSIONS: We identified three new miRNAs predictive of distant relapse in operable SCC. Future miRNA studies should account for differences between NSCLC subtypes.

Molecular profiles of non-small cell lung cancers in cigarette smoking and never-smoking patients.

PURPOSE: Molecular features of non-small cell lung cancer (NSCLC) in never-smokers are not well recognized. We assessed the expression of genes potentially related to lung cancer etiology in smoking vs. never-smoking NSCLC patients. METHODS: We assayed frozen tumor samples from surgically resected 31 never-smoking and 54 clinically pair-matched smoking NSCLC patients, and from corresponding normal lung tissue from 27 and 43 patients, respectively. Expression of 21 genes, including cell membrane kinases, sex hormone receptors, transcription factors, growth factors and others was assessed by reverse transcription - quantitative PCR. RESULTS: Expression of 5 genes was significantly higher in tumors of non-smokers vs. smokers: CSF1R (p<0.0001), RRAD (p<0.0001), PR (p=0.0004), TGFBR2 (p=0.0027) and EPHB6 (p=0.0033). Expression of AKR1B10 (p<0.0001), CDKN2A (p<0.0001), CHRNA6 (p<0.0001), SOX9 (p<0.0001), survivin (p<0.0001) and ER2 (p=0.002) was significantly higher in tumors compared to normal lung tissue. Expression of AR (p<0.0001), EPHB6 (p<0.0001), PR (p<0.0001), TGFBR2 (p<0.0001), TGFBR3 (p<0.0001), ER1 (p=0.0006) and DLG1 (p=0.0016) was significantly lower in tumors than in normal lung tissue. Expression of IGF2 was higher in tumors than in healthy lung tissue in never-smokers (p=0.003), and expression of AHR (p<0.0001), CSF1R (p<0.0001) and RRAD (p<0.0001) was lower in tumors than in healthy lung tissue in smokers. CONCLUSION: Expression of several genes in NSCLC is strongly related to smoking history. Lower expression of PR and higher expression of ER2 in tumors suggests a possibility of hormonal therapeutic intervention in selected NSCLC patients. Distinct molecular features of NSCLC in never-smokers, e.g. CHRNA6 upregulation, may prompt new treatment strategies.

Determination of sentinel lymph node (SLN) status in primary breast cancer by prospective use of immunohistochemistry increases the rate of micrometastases and isolated tumour cells: analysis of 174 patients after SLN biopsy.

AIM: The objective of the present study was to evaluate the prospective use of immunohistochemistry (IHC) for histopathological diagnosis of sentinel lymph node(s) (SLN) in primary breast cancer using stage migration and non-SLN metastases as endpoints in relation to metastatic involvement. METHOD: Serial sectioning and prospective use of IHC were applied to SLN examination in addition to routine haematoxylin-eosin staining in 174 consecutive patients with unifocal T1-T2 breast cancer included in a National Sentinel Node Study. Axillary lymph node dissection (ALND) was performed in all cases with macrometastases, micrometastases and isolated tumour cells (ITC). RESULTS: The SLN was found in 173/174 patients and a metastatic foci was found in 50 patients including 28/50 with macrometastases, 16/50 with micrometastases and 6/50 with ITC. IHC detected 3/16 of the micrometastases and 4/6 of ITC. Stage migration from N0 to N1mi was encountered in 3/132 patients by use of IHC. Non-SLN metastases were noted in 15/28 of patients with macrometastases and in 3/16 of patients with micrometastases, whereas no patient with ITC had additional metastases (p=0.007). CONCLUSION: The prospective use of IHC and serial sectioning for histopathological diagnosis of SLNs increased the detection rate of N1mi and ITC, but only 3/132 patients were stage-migrated by use of IHC. Patients with ITC did not have any risk of non-SLN metastases, supporting that ALND can safely be omitted in this group of patients.

Early complete donor hematopoietic chimerism in peripheral blood indicates the risk of extensive graft-versus-host disease.

Achievement of complete donor hematopoietic chimerism (CC) is the goal of allogeneic stem cell transplantation (allo-SCT). Persistence of recipient hematopoiesis augments the risk of relapse, which is one of the main reasons for mortality after allo-SCT. Another main reason for morbidity and mortality is severe extensive chronic graft-versus-host disease (cGvHD). We examined chimerism in peripheral blood of 54 allogeneic stem cell recipients using multiplex STR-PCR method and compared it with the timing and severity of cGvHD. In total, 25 patients achieved early CC (by day 100 post transplant) at a median time of 60 days. In total, 21 of them developed extensive cGvHD. In those patients CC uniformly preceded emergence of cGvHD by a mean of 85 days. A total of 26 patients obtained late CC at a median time of 270 days post transplant. Of this group, only eight patients developed extensive disease. Development of cGvHD in those patients preceded achievement of CC in 10 of 13 cases by a mean of 100 days. The difference between early and late CC groups as to the frequency of the extensive cGvHD was statistically significant (P<0.001). Also, there was a significant correlation of the time of CC and time between CC and cGvHD. Additionally, patients with early CC developed significantly more severe cGvHD measured by the need of three-drug treatment to control the disease (P<0.005). It can be concluded that achievement of early complete donor hematopoietic chimerism in peripheral blood is strongly predictive of severe extensive GvHD.

First Polish DNA "manhunt"--an application of Y-chromosome STRs.

This study presents the application of Y-chromosomal STR polymorphisms to male identification in the case of a serial rapist and woman murderer in Poland. Since August 1996 a rapist from Swinoujscie (northwest Poland) committed at least 14 rapes. In the year 2000 he brutally raped 8 young girls and murdered a 22-year-old girl. DNA profiles obtained from semen stains left at the scenes of crime gave information that one and the same man had committed all the rapes. The Y-chromosome haplotype (9 loci) obtained was used for the elimination process of 421 suspects. One man was found who had an identical DNA profile in all Y-chromosome STR loci analysed and possessed common alleles in 9 out of 10 autosomal loci, strongly suggesting that the real rapist and the typed man were closely related males. Analysis of reference DNA obtained from the man's brother revealed an identical DNA STR profile to that identified at the crime scenes. To the best of our knowledge this is the first case in Poland and probably in Eastern Europe where DNA typing of a large population was used to identify the offender.

[Population genetics of 9 Y-chromosome STR loci w Northern Poland]. / Genetyka populacyjna 9 loci typu Y-STR w populacji Polski Pólnocnej.

A total of 508 unrelated males from the North Poland population were analyzed for 9 Y-chromosome STRs (DYS 19, DYS 390, DYS 393, DYS 392, DYS 391, DYS 389I, DYS 389II and DYS 385I/II) using two multiplex reactions and detection of PCR products using capillary electrophoresis. In the analyzed sample 328 different haplotypes were identified, among which 264 were unique. It was found that the model for a Polish population haplotype (DYS 19*17, DYS 390*25, DYS 393*13, DYS 392*11, DYS 391*10, DYS 389I*13, DYS 389II*30, DYS 385I/II*10,14) is almost 15 times more frequent in our population than in a cumulative European one. The haplotype diversity/discrimination index calculated for 9 loci is 0.9943. In the analysed population sample three mutations were detected in the DYS19 (duplication) and DYS385I/II loci (triplications).

[Application of pcr in paternity determination in pregnancy preceded by rape]. / Zastosowanie reakcji lancuchowej polimerazy (PCR) we wczesnej ciazy w badaniu spornego ojcostwa zwiazanego z gwaltem.

Authors present a case of PCR (Polymerase Chain Reaction) technique application for the paternity determination in pregnancy preceded by the rape. Diagnostic material was obtained in the 10th week of pregnancy by the use of transabdominal Chorionic Villous Sampling under the echo-guidance and its DNA feature was compared against the DNA material obtained from the vagina after the rape and the material from mother and her husband. The PCR technique revealed in chorionic villi the presence of allele that were present in material deriving from mother and her husband as well as no even single allele from the material of violator. Exclusion of violator as a father of the foetus effected in decision of continuing the pregnancy.

Forensic validation of a multiplex containing nine STRs--population genetics in northern Poland.

This paper presents the allele frequency distributions for the nine loci (D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820) present in the commercially available Profiler Plus kit. DNA samples of 202 individuals from Northern Poland were amplified in a multiplex reaction with subsequent automatic detection using capillary electrophoresis. All loci met the Hardy-Weinberg equilibrium conditions. The calculated probability of identity was 2.26 x 10(-11) giving an average probability of identity of 1 in 44 billion. Considerable improvement of analysis precision was observed after substituting the GeneScan 500 for the fluorescent ladder 60-400 bp. The influence of DNA concentration on stutter and artefact formation and the ratio of heterozygote alleles was analysed.

Y-chromosomal polymorphic loci DYS19, DYS390, DYS393 in a population sample from northern Poland.

Short tandem repeats (STR) are presently of particular use in the field of forensic science, evolution and anthropology. Y-chromosomal STR systems are widely used for population genetics, population history, and for male identification in forensic cases. Here we present an examination of DYS19, DYS390 and DYS393 allele frequencies in a northern Polish population sample. The calculated indices reflect the potential of these markers for application in forensic casework. Statistically significant differences were observed between most western European populations and the Polish cohort when comparing homogeneity of distribution of DYS19 and DYS390 alleles. For three analyzed loci in a sample of 176 males, 43 haplotypes were observed. The studies revealed some rare alleles and a new allele, allele 16, in the DYS393 system. Sequencing by capillary electrophoresis (PE ABI 310) showed the presence of 16 GATA repetitive elements, confirming results obtained after capillary electrophoresis of the DNA fragment. Additionally, sequencing revealed the presence of a novel transversion (A->C) in the analyzed sample.

Genetic determinants of plasma ACE and renin activity in young normotensive twins.

OBJECTIVE: We investigated the determinants of plasma renin activity (PRA) and plasma levels of angiotensin-converting enzyme (pACE), including the effect of the D/I polymorphism of the angiotensin-converting enzyme (ACE) gene, in monozygotic (MZ) and dizygotic (DZ) twins. METHODS: Sixty-nine pairs of twins underwent measurements of blood pressure, pACE and ACE D/I genotyping. In addition, in 30 pairs ambulatory blood pressure (ABP) monitoring was carried out. To ascertain twin's zygosity, some highly discriminating variable number of tandem repeats micro- and mini-satellite systems were analysed by polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis and silver staining. The D/I polymorphism was assessed by PCR; pACE was measured in triplicate with a colorimetric assay, and PRA by a commercial kit. In DZ twins, identity by descent of the D/I alleles was examined by PCR amplification of a highly polymorphic simple sequence repeat at the human growth hormone gene. RESULTS: pACE levels were significantly (P < 0.01) higher in DD (9.27 +/- 2.60 IU/l, mean +/-SD) than in II (6.68 +/- 3.0), with DI having intermediate levels (7.93 +/- 2.7). No difference of PRA between different D/I genotypes was found. Twin data analysis showed a statistically significant heritability of pACE, but not of PRA. No differences between MZ and DZ twins in PRA, pACE and the relationship of the D/I genotype with pACE was found. Besides showing that the D/I genotype was the most important predictor of pACE, a multivariate analysis demonstrated that identity by descent of the D/I allele, as assessed by growth hormone (GH) genotyping, also significantly affected pACE. CONCLUSIONS: In this study of normotensive twins, pACE and not PRA showed significant heritability, the former being tightly associated with the D/I ACE gene polymorphism, and/or with a quantitative trait locus in linkage disequilibrium with it.

Heritability of plasma leptin levels: a twin study.

OBJECTIVE: To examine the influence of genetic factors on plasma leptin levels. SUBJECTS AND METHODS: We measured plasma leptin levels, body mass index and body fat distribution in healthy young female monozygotic (n = 19) and dizygotic (n = 14) twins. The twin zygosity was verified by determination of short tandem repeat and amplified fragment length polymorphism systems. The genetic analysis included analysis of variance-based and maximum likelihood-based methods. RESULTS: Plasma leptin levels were correlated significantly with body mass index (r = 0.59, P < 0.001), waist circumference (r = 0.54, P < 0.001) and hip circumference (r = 0.63, P < 0.001), but not with age (r = -0.17) or the waist:hip ratio (r = 0.02). The heritability estimates derived from intraclass correlations were significant for body mass index (P = 0.001), waist circumference (P = 0.004), hip circumference (P = 0.01) and plasma leptin levels (P = 0.005), but not for the waist:hip ratio (P = 0.22). In the maximum likelihood-based path analysis, heritability was estimated at 79% for body mass index and at 73% for plasma leptin levels. After adjustment for body mass index, the heritability estimate for leptin levels from the model-fitting approach was 55%. CONCLUSIONS: Genetic factors are major determinants of plasma leptin levels in humans and may account for as much as half of the variance in leptin levels.

HUMFIBRA allele distribution in northern Poland using capillary electrophoresis.

Amplification products of the HUMFIBRA locus were analysed in a population from Northern Poland using capillary electrophoresis. The most frequent alleles were 21, 22 and 20 with a frequencies of 0.201, 0.179 and 0.167 respectively, and five rare variants were observed with frequencies less than 0.015. A homogeneous allele distribution was found between Polish and Dutch populations and significant differences between Poles and Italians. The high discrimination power (0.963), polymorphic information content (0.849) and heterozygosity confirm the usefulness of HUMFIBRA locus for forensic identification purposes. The application of capillary electrophoresis offers the possibility of precise identification of rare variants.

[Polymorphism of the ACE gene and left ventricular morphology and function in patients with borderline, mild and moderate hypertension]. / Polimorfizm genu ACE a morfologia i funkcja lewej komory serca u chorych z nadcisnieniem tetniczym granicznym, lagodnym i umiarkowanym.

It has been reported that the allel D of an insertion/deletion (I/D) polymorphism of the angiotensin I converting enzyme (ACE) gene is associated with the conditions of increased cardiovascular risk, including the left ventricular hypertrophy and the dysfunction. We examined the relation between the genotype of ACE gene and the left ventricular function in normotensives and in borderline, mild and moderate hypertensives. We investigated 128 subjects, 47 first-diagnosed untreated hypertensives and 81 normotensives. The M-mode and Doppler echocardiography were used to quantify LV mass and function. The insertion/deletion ACE polymorphism was identified using polymerase chain reaction. Left ventricular indexes of the morphology and function were analyzed. We compared ambulatory blood pressure profiles between all genotypes in both groups. There were no significant differences in indexes of the left ventricular hypertrophy in studied normotensives and borderline to mild hypertensives. Our results indicate that allel I might be associated with selected parameters of diastolic function, while allel D with selected parameters of systolic function, of the left ventricle. Results of this study suggest also probable relation between allel D and variability of the diastolic arterial pressure in both investigated groups.

Ambulatory systolic blood pressure is related to the deletion allele of the angiotensin I converting enzyme gene in young normotensives with parental history of hypertension.

The aim of the present study was to evaluate the relationship between the angiotensin I converting enzyme (ACE) gene polymorphism and ambulatory blood pressure in young normotensive males with (n=45) and without (n=100) family history of hypertension. Twenty-four hour and daytime systolic blood pressure was significantly higher in subjects with a parental history of hypertension. Ambulatory blood pressure values did not differ significantly across ACE genotypes in subjects with negative family history of hypertension. In subjects with a parental history of hypertension, there was a significant positive association between the D allele of the ACE gene polymorphism and 24-h, daytime and nighttime systolic blood pressure. For twenty-four hour systolic blood pressure there was an average 9 mmHg difference between subjects with DD and II genotypes. The results indicate that in normotensive subjects with a genetic predisposition to hypertension, ambulatory systolic blood pressure is related to the D allele of the ACE gene.

No effect of genetic variation at the angiotensinogen locus on ambulatory blood pressure level in normotensive subjects.

The aim of the study was to evaluate the potential association between ambulatory blood pressure and the molecular variants T174M and M235T of the angiotensinogen gene in a random sample of young normotensive men (n = 145). The two point mutations were detected using restriction digests of a mispairing polymerase chain reaction product. Twenty-four-hour ambulatory blood pressure monitoring was performed with a SpaceLabs 90207 device. Ambulatory blood pressure levels did not vary according to T174M and M235T genotypes. When the subjects were grouped according to their blood pressure level (as indicated by tertiles of their 24-h ambulatory blood pressure), no significant differences in allele frequencies between the three groups were found. Our results indicate that the T174M and M235T molecular variants of the angiotensinogen gene have no major influence on ambulatory blood pressure in young normotensive subjects.

Genetic influences on insulinemia in normotensive twins.

The aim of this study was to establish the contribution of genetic factors to the variance of plasma insulin concentration in healthy, normotensive twins. Seventeen pairs of monozygotic (MZ) and 17 pairs of dizygotic (DZ) twins were investigated. The test of genetic variance revealed a significantly larger within-pair variance of fasting plasma insulin (FPI) and a relative insulin resistance (RIR) in the DZ twins, in comparison with the MZ twins. Both FPI and RIR had a higher intraclass correlation coefficient in the MZ twins than in the DZ twins; the corresponding heritability estimates were 0.54 for FPI and 0.66 for RIR. Adjusting for age, gender, and body mass index did not affect heritability estimates for either FPI or RIR. Our data indicate that genetic factors are important determinants of insulinemia in normal subjects, independent of body mass index.

Frequencies for five short tandem repeat (STR) systems in a population from north Poland.

A population study of unrelated individuals from North Poland (Gdansk area) was carried out to investigate the allele distributions of the five STR systems HUMCD4, HUMFES/FPS, HUMVWA31, HUMTH01 and ACTBP2. PCR products were separated on horizontal non-denaturing polyacrylamide gels followed by silver staining. For all STR systems analysed the distribution of observed phenotypes did not deviate from Hardy-Weinberg equilibrium. A comparison of allele distributions between Polish and other European Caucasian population samples is presented.

Drastically decreased transcription from CII-activated promoters is responsible for impaired lysogenization of the Escherichia coli rpoA341 mutant by bacteriophage lambda.

It was demonstrated previously that a mutation, rpoA341, in the gene encoding the alpha subunit of Escherichia coli RNA polymerase prevents lysogenization by bacteriophage lambda. The rpoA341 allele is known to be responsible for impaired transcription of some positively regulated E. coli chromosomal operons. Here we demonstrate that the inhibition of lysogenization of the rpoA341 mutant is a result of drastically decreased transcription from positively regulated phage promoters. We were unable to detect any transcripts originating from the CII-activated pE, pI and paQ promoters (important for lysogenic development) in the phage-infected rpoA341 mutant, in contrast to an otherwise isogenic rpoA+ strain. The results are discussed in the light of other reports showing that activation of the pE promoter by CII protein in vitro is decreased only about fivefold when the native alpha subunit is replaced by truncated alpha polypeptides.

Population studies of three AMPFLPs systems in a north Polish population.

Allele and phenotype frequencies for D1S80, D17S5 and ApoB were determined in a population sample of more than 200 unrelated persons from North Poland using the PCR method. For D1S80, D17S5 and ApoB 19, 13 and 21 alleles respectively were observed. No deviations from Hardy-Weinberg equilibrium were detected. All three systems have discrimination values above 92% and a cumulative discrimination index of 4.5 x 10(-4).

[Identification analysis using PCR of fresh or fixed and paraffin wax embedded tissue]. / Badania identyfikacyjne metoda PCR tkanek swiezych oraz utrwalonych i zatopionych w blokach parafinowych.

The aim of the work was a selection of the best method of DNA extraction from soft tissues, and than application of this method to the fixed and paraffin wax embedded tissue. Isolated DNA was amplified using PCR method. Amplification was done for amelogenin locus (sex specific sequences) and some polymorphic systems like mini- and microsatellites. It was found that it is possible to amplify 788 and 977 bp long amelogenin fragments, using DNA isolated from fixed and paraffin wax embedded tissue stored for two years. The case of identification of unknown person (victim of fire at Gdansk Shipyard show room) on basis of DNA isolated from soft tissue was presented.