Optimization of the method of RNA isolation from paraffin blocks to assess gene expression in breast cancer.

UNLABELLED: Molecular oncology increasingly needs the assessment of tumor gene expression profile (transcriptome), most commonly by determination of RNA-based molecular markers employing the technique of quantitative real-time polymerase chain reaction (Q-PCR). However, as all are methods based on RNA, to date, the experience in Q-PCR is mostly limited to freshly collected material frozen at -80 degrees C, i.e. showing no signs of RNA degradation. The aim of the present study was to implement into practice a method of RNA isolation from formalin-fixed and paraffin-embedded (FFPE) breast carcinoma samples collected during routine surgical and histopathological procedure, to further employ it in expression analysis by Q-PCR. The RNA isolation kit RNeasy FFPE (QIAGEN) was used. It was demonstrated that in samples subjected to DNAse digestion, the mean concentration of the obtained RNA was low (46 ng/microl), while during the isolation performed using solely gDNA Eliminator columns, the authors obtained RNA with an almost fourfold higher concentration value. A comparison was made between isolation effectiveness using varying amounts of input material. It was noted that isolation efficacy was lower when three sections were employed (the concentration value of 178 ng/microl) as compared to 5-8 sections (279 and 302 ng/microl, respectively). RNA quality assessment was also performed employing the method of capillary electrophoresis by the "lab-on-a-chip" technology of Agilent Bioanalyzer 2100. Freshly prepared material yielded in single cases samples containing RNA18S and RNA28S populations, while in samples isolated from archival paraffin blocks, the obtained RNA showed more considerable degradation, thus, was of lesser quality. In the analysis of 20 samples from the second collected series, the majority of samples were characterized by the RNA Integrity Number (RIN) values in the range of 2-2.5, still indicative of a substantial degree of RNA degradation. The mean isolation effectiveness in the second series was 885 ng/microl. In 10 of 20 blocks isolated, we succeeded in obtaining sufficient RNA concentration, above 500 ng/microl. It was also noted that the storage time did not affect the amount of RNA obtained from a block: while isolating RNA from freshly prepared blocks, we achieved similar concentrations as when analyzing the archival material. CONCLUSIONS: the key in preserving RNA quality in paraffin blocks is the timing of material collection and fixing. Routine paraffin blocks allow for obtaining RNA for molecular studies, yet with features of considerable degradation.

Spermatocyte-specific expression of constitutively active heat shock factor 1 induces HSP70i-resistant apoptosis in male germ cells.

Spermatocytes, the most sensitive male germ cells to heat-induced apoptosis, do not respond to hyperthermia by inducing heat shock proteins (HSPs), including HSP70i, which has been previously shown to confer resistance to apoptosis in somatic cells. To dissect the mechanism of heat-induced apoptosis and to determine if we could protect spermatocytes by expressing HSP70i, we engineered transgenic mice that express in spermatocytes constitutively active heat shock transcription factor (HSF)1. Such HSF1 expression did not lead to transcription of inducible Hsp70 genes, but instead induced caspase-dependent apoptosis that mimicked heat shock-induced death of spermatogenic cells. Both mitochondria-dependent and death receptor-dependent pathways appear to be involved in such HSF1-induced apoptosis: the levels of Bcl-2 family proteins became increased, p53 protein accumulated and expression levels of caspase-8 and death-receptor-interacting proteins (including Fas-associated death domain protein and TNF receptor associated death domain protein) became elevated. Surprisingly, the constitutive spermatocyte-specific expression of HSP70i in double-transgenic males did not protect against such HSF1-induced apoptosis.

Association of polymorphism of LTalpha and TNF genes with Graves' disease.

Graves' disease (GD) is an autoimmune disease, which develops on the basis of an interaction between genetic, environmental and endogenous factors. GD is associated with some HLA genes. Closely linked with them are TNF genes (TNF and LTalpha). Their role in the pathogenesis of GD is still unclear. Two functional polymorphisms within TNF genes include a substitution of G with A in intron I of LTalpha gene and the same one at position -308 in the TNF gene promoter. We carried out a case-control study for the analysis of the contribution of TNF genes to GD in Polish patients. 156 patients with GD diagnosed by clinical data were investigated and compared to 80 healthy persons with negative familial anamnesis. Both TNF and LTalpha were analysed by PCR/Nco I RFLP. The allelic frequency of the rarer TNF2 (A) allele, was 24.7% in GD patients, significantly higher than in healthy persons (9.3%; p<0.0001). The OR was 4.38 for this allele. The frequency of heterozygotes was 41.8% in GD, as compared to 13.6% in the control group. The allelic frequency of the rarer LTB*1 (G) allele was also significantly increased: from 21.9% in the control group to 37.2% in GD patients (p<0.01; OR 2.81). The frequency of heterozygotes was 48.7% in GD, and 28.8% in the control group. The results indicate that TNF genes may contribute to GD in the Polish population.

[Differential criteria between papillary and follicular thyroid carcinoma--initial conclusions from a multicenter trial]. / Kryteria róznicowania miedzy rakiem brodawkowatym i pecherzykowym tarczycy--pierwsze wnioski z badania wieloosrodkowego.

Histopathological diagnosis of thyroid cancer is difficult and requires much experience. Pathologists have to know many histopathological variants and be aware of the current diagnostic criteria. The aim of the study was to unify criteria applied all over the country and compare whether the accuracy of diagnosis has changed in the course of the last fifteen years. In a multicenter trial, 36 pathologists from 25 centers reevaluated 232 thyroid tumors operated between 1985-1998. The reference diagnosis was given on the basis of evaluation made by four experienced pathologists. The two-step analysis was performed. At first, the accuracy of the diagnosis of malignant neoplasm was evaluated. Then, the accuracy of the diagnosis of the cancer histotype was analyzed, with estimation of kappa coefficients and their asymptomatic standard error. Comparison of primary and reference diagnoses revealed statistically significant differences--in 17% of cases the primary diagnosis of cancer was not confirmed by experienced pathologists. Kappa coefficient for the diagnosis of cancer histotype was 0.53 + 0.06. On the contrary, the diagnoses made by the participants of the trial did not differ significantly from the reference ones. Kappa coefficient for the diagnosis of cancer histotype was significantly higher than for primary diagnoses with 0.63 +/- 0.10 (p < 0.001). The first results of the multicenter trial indicated that the most frequent diagnostic error made at primary diagnosis was the overdiagnosis of follicular thyroid carcinoma. Thus, a summary of strict criteria for papillary and follicular thyroid carcinoma is also given.

[Criteria for histopathologic diagnosis of thyroid cancer in a quick test of accuracy]. / Kryteria rozpoznania histopatologicznego raka tarczycy w szybkim tescie zgodnosci.

A quick test of accuracy of histopathological diagnosis of thyroid carcinoma was performed in May 2000 during the meeting of Polish thyroid cancer group (Committee for Epidemiology, Diagnosis and Treatment of Thyroid Carcinoma). 29 pathologists participated in the test and evaluated 8 cases of thyroid carcinoma and 14 benign thyroid lesions. All cases were chosen from the current material sent for pathologic evaluation to the Institute of Oncology in Gliwice due to diagnostic difficulties. In total, 591 diagnoses were made and were the subject of the presented analysis. They were compared with reference diagnosis in two aspects. First, the accuracy of the distinction between malignant and benign lesions was evaluated. 72.5% of diagnoses were concordant with the reference. The false diagnosis of cancer in a benign lesion was observed 133 times (22.5% of all diagnoses). A reverse error--a false exclusion of cancer--was seen in 29 diagnoses (4.9%). Chi 2 test revealed a statistically significant difference between the participants' diagnoses and reference ones (p < 0.0001). Overdiagnosis of cancer was the most frequent at the diagnosis of follicular or oxyphilic cancer. With reference to the diagnosis of cancer histotype, concordant diagnoses were seen in 40-47% of cases with the lowest accuracy of the diagnosis of oxyphilic (40% of correct diagnoses) and follicular (50%) cancer. The causes of false diagnoses may be divided in two groups: sample-related causes (sampling of surgical specimens, lack of standard description, insufficient number of samples, poor quality of staining) and diagnostic errors: non-compliance with diagnostic criteria and inappropriate setting of diagnoses, which require immunohistochemical confirmation.

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.

Gene expression profile analysis in laryngeal cancer by high-density oligonucleotide microarrays.

The assessment of gene expression profile in laryngeal cancer shall allow to implement molecular biology methods in diagnostics, as well as in prognosis of the course of disease. Thus, it may influence the choice of the most optimal decisions in regards to the method of treatment, extent of surgical procedure, or the necessity of adding post-operative radiotherapy. The aim of the project was to analyse the gene expression profile of laryngeal cancer using oligonucleotide microarrays, aiming to derive novel molecular markers for that carcinoma. The study comprised a group of 14 patients (12 males and 2 females) with squamous cell laryngeal carcinoma, diagnosed and surgically treated between 2005 - 2007 in the ENT Department of the Silesian Medical University in Katowice, Poland. RNA was isolated from frozen tissue fragments. To assess gene expression profile, high density oligonucleotide microarrays (Affymetrix U 133 Plus 2.0) were applied, with over 54 thousand probesets for over 47 thousand transcripts. Four genes, previously not assesed in diagnostic context in laryngeal carcinoma, seemed to be valuable markers of that neoplasm. These are: metalloproteinase ADAM12, cycline-dependent kinase 2 - CDK2, kinesine 14 - KIF14, suppressor 1 of checkpoint - CHES1.