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.

Understanding variations in survival for colorectal cancer in Europe: a EUROCARE high resolution study.

BACKGROUND: Marked differences in population based survival across Europe were found for colorectal cancers diagnosed in 1985-1989. AIMS: To understand the reasons for these differences in survival in a new analysis of colorectal cancers diagnosed between 1988 and 1991. SUBJECTS: A total of 2720 patients with adenocarcinoma of the large bowel from 11 European cancer registries (CRs). METHODS: We obtained information on stage at diagnosis, diagnostic determinants, and surgical treatment (not routinely collected by CRs) and analysed the data in relation to three year observed survival, calculating relative risks (RRs) of death and adjusting for age, sex, site, stage, and determinants of stage. RESULTS: Three year observed survival rates ranged from 25% (Cracow) to 59% (Modena), and were low in the Thames area (UK) (38%). Survival rates between registries for "resected" patients varied less than those for all patients. When age, sex, and site were considered, RRs ranged from 0.7 (95% confidence intervals (CI) 0.6-0.9) (Modena) to 2.3 (95% CI 1.9-2.9) (Cracow). After further adjustment by stage, between registry RR variation was between 0.8 (95% CI 0.6-0.9) and 1.8 (95% CI 1.5-2.2). Inter-registry RR differences were slightly reduced when the determinants of stage (number of nodes examined and liver imaging) were included in the model. The reduction was marked for the UK registries. CONCLUSIONS: The wide differences across Europe in colorectal cancer survival depend to a large extent on differences in stage at diagnosis. There are wide variations in diagnostic and surgical practices. There was a twofold range in the risk of death from colorectal cancer even after adjustment for surgery and disease stage.

[Monitoring of bone metastases]. / Monitorowanie leczenia przerzutów nowotworowych do kosci.

Current knowledge about an incidence of bone metastases, use of bisphosphonates and assessment of response to the treatment are surveyed. The bone metastases are quite frequent in patients with breast and prostate cancers. High doses of intravenous pamidronate are particularly useful in the treatment of these patients. Prior to therapy with bisphosphonate special score elaborated by RE Coleman should be calculated. The new biochemical bone resorption markers especially Ntx i Crosslaps seems to be the most efficient for evaluation metastases response.

[Single sigmoidoscopy for screening of colorectal cancer]. / Jednorazowa sigmoideoskopia w badaniach przesiewowych raka jelita grubego.

Once-only sigmoideoscopy of person aged 57 is successful and safe method of screening of colorectal cancer. However acceptance of invitation to screening was about 25%. Because of that we postulated once-only sigmoideoscopy screening for patients aged 57 hospitalized from other than digestive tract neoplasms causes.

[Current possibilities for the decrease of mortality risk in patients with head and neck cancers]. / Obecne mozliwosci zmniejszenia ryzyka zgonu na nowotwory glowy i szyi.

The frequency, survival rates and risk factors for head and neck cancer were reviewed. The most common site in Poland is larynx, accounting for almost 50% of head and neck sites. The second group (each about 15%) are neoplasms of mouth and lip. The differences in 5-year survival for males and females with cancer of larynx, pharynx and mouth are not significant. Our survival rates are not significantly different from European mean. The main risk factors for head and neck cancer are smoking and alcohol drinking together with rare consumption of fruit and vegetables. Dentists are particularly responsible for early diagnosis of head and neck cancer. Chemioprevention can decrease the risk of second primary cancer and cytogenetic tests should in future decide about primary and secondary prevention.

[Survival of patients with the most frequent malignant neoplasms in the population of Krakow in the years 1985-1989]. / Przezycia chorych na najczestsze nowotwory zlosliwe w populacji Krakowa w latach 1985-1989.

In 1985-1989 there was not significant improvement of survival rates for the most common cancer sites in Cracow population. Survival of Cracow patients with colorectal, breast and larynx cancer and chronic lymphatic leukaemia were significantly below European mean. This situation is probably typical for whole Polish population.

Evaluation of certain risk factors for lung cancer in Cracow (Poland)--a case-control study.

The relationship of lung cancer risk to cigarette smoking, occupational exposure, air pollution at permanent residence and usual frequency of consumption of fruits and vegetables was analysed in a case control study in Cracow, Poland. The cases were 176 male lung cancer patients diagnosed in 1992-1994 with histological confirmation of the diagnosis, obtained from the population-based Cracow Cancer Registry, and 341 controls randomly selected from the general population. Classification of exposure to outside air pollution was based on measured level of total suspended particular matter and sulphur dioxide by particular districts of town (from 1973 to 1980). Except calculation of odds ratios for male lung cancer associated with exposures among all study subjects, the association between lung cancer risk and frequency of consumption of fruit and vegetables was examined among drinkers of vodka above average. Risk of lung cancer was increased significantly with increasing number of pack-years of smoking (OR = 18.7 for more than 40 pack-years). The significant inverse association of frequent usual consumption of boiled vegetables was present both among all subjects and among vodka drinkers (OR = 4.6 and 12.5, respectively, for a rare consumption). The risk of male lung cancer was negatively associated with the level of air pollution but positively with the percentage of occupationally exposed. Our study provides no evidence of a significant harmful effect of air pollution and found a strong inverse association between frequent fruit and vegetables consumption and lung cancer risk.

Dietary factors and risk of prostate cancer in Poland. Results of case-control study.

The relationship between cigarette smoking, vodka drinking and consumption of 44 food items typical of the Polish diet were analyzed in a case-control study performed in Cracow, Poland, among 76 cases of prostate cancer and 152 controls. Cigarette smoking and drinking of vodka were not significantly influencing the prostate cancer. The men who ate smoked or fried fish or liver at least once per week had almost half of the risk of prostate cancer of the men who ate those food rarely.

[Ovarian cancer and selected life style habits]. / Rak jajnika a wybrane elementy stylu zycia.

Analysis of population based case-control study performed in Cracow Poland in 1988-1990 on 81 cases of histologically proven epithelial ovarian cancer and 162 age matched controls have shown that frequent consumption of legumes was associated with significantly decreased risk. Smoking and drinking of vodka were not significant related to ovary cancer risk.

[Second primary malignant neoplasms in patients treated radically for carcinoma of oral cavity, larynx and lungs]. / Drugie pierwotne nowotwory zlosliwe u chorych leczonych radykalnie z powodu raka jamy ustnej, krtani i pluca.

About 1% of the patients with oral cavity, larynx, and lung cancer get second primary malignant neoplasms every year. For laryngeal cancer the percentage exceeds 10. Second primary tumors in oral cavity, larynx, and lung cancer patients are also tobacco-related neoplasms. Smoking cessation by patients with oral cavity, larynx, and lung cancer may have significant influence on the prognosis. Vitamin A (retinol) and its analogues are promising for human cancer prevention.

Survival studies: Cracow Cancer Registry experience.

The experience of the Cracow Cancer Registry is presented to show how useful a population-based survival study can be. Survival rates for lung, breast, and colorectal cancer are compared over two periods of time. In addition, the survival experience of patients treated at the Oncologic Center in Cracow is compared with the U.S. SEER data.

Breast cancer and smoking, vodka drinking and dietary habits. A case-control study.

The relationship between cigarette smoking, vodka drinking and consumption of 44 food items typical of the Polish diet were analysed in a case-control study in Cracow, Poland, among 127 cases of breast cancer and 250 controls randomly selected from the general population. Cigarette smoking was not significantly influencing the breast cancer risk. Compared with never-drinkers, the habit of vodka drinking 20 years earlier significantly increased breast cancer risk in women below 50 years of age (multivariate OR was 4.4 with 95% CI 1.6-12.4). Frequent consumption of boiled vegetables 20 years earlier (greater than 3 times per week) was associated with a decreased risk of breast cancer in women aged 50 years and more (multivariate OR was 0.4 with 95% CI 0.2-0.8).

[Selected aspects of diagnosis and treatment of prostatic cancer]. / Wybrane aspekty diagnostyki i terapii raka stercza.

Prostatic cancer is one of the most frequent malignant tumours in men. The chronic course of the disease and the opportunity of selecting of one of many available treatment methods make possible adjustment of the management to the individual problems of the patient. This leads, however, to various difficulties connected with the selection of optimal management in various clinical situations. The purpose of the paper was presentation of selected problems in the diagnosis and treatment of prostatic cancer in the light of own clinical experience, in the first place.

[Five year survival rate in patients with malignant neoplasms treated for the first time in the Oncology Center, M. Sklodowska-Curie Institute, Cracow Branch in the years 1979-1981]. / Przezycia 5-letnie chorych na nowotwory zlosliwe leczonych po raz pierwszy w Centrum Onkologii--Instytucie im. M. Sklodowskiej-Curie, Oddzial w Krakowie, w latach 1979-81.

In the light of data from the admission register of the Cracow Branch of the Oncology Centre percent values are presented of actual 5-year survival rate of patients treated for the first time in the period 1979-1981. The group of neoplasms with relatively good prognosis (i.e. survival rate for 5 years over 50%) included six sites: skin, lip, endometrium and cervix, larynx, and Hodgkin's disease. The group of poor prognosis (5-year survival rate below 30%) included: cancer of the kidney, nasopharynx and oropharynx, nasal cavity and nasal sinuses, tongue, rectum, prostate, bladder, lungs, stomach and oesophagus, and brain tumours. The present situation was compared with data from the preceding years and with the survival rates in the populations of Cracow and the USA.

[Predicting the incidence of malignant neoplasms in Cracow in the year 2000]. / Nowotwory zlosliwe w miescie Krakowie w perspektywie roku 2000.

In connection with the data of the Cracov Tumor Registry concerning the 1970-1985 period and the population prognoses of the city of Cracov the expected incidence and the number of patients with most common malignant neoplasms in the year 2000 has been estimated. One can foresee the continuance of the decrease of the risk and incidence of malignant neoplasms of the stomach in both sexes and of cancer of the lung and breast in women. The incidence of malignant neoplasms in general as compared with the year 1985 will in the year 2000 increase by ca. 60% in men and by ca. 39% in women.

[Comparison of 5-year survival rates of patients with the most common malignant neoplasms living in urban and rural areas in the years 1982-1983]. / Porównanie przezyc 5-letnich chorych na najczestsze nowotwory zlosliwe zamieszkalych w miescie i na wsi w latach 1982-1983.

Almost every fifth man and every third woman with malignant neoplasm living in urban and rural areas of the Cracov region, had in the years 1982-1983 the chance to survive 5 years. 5 year survivals of patients from urban and rural areas did not differ significantly. Also in other periods of time between 1976-1977 and 1982-1983 the differences were insignificant. As compared with data taken from other populations in the years 1982-1983, particularly low level of 5 year survivals has been noted in Cracov of male patients with cancer of the colon and in women with cancer of the uterine body. The survivals of patients with malignant neoplasms of the colon in both sexes, of the prostate and kidney in men and of breast in women are strikingly low in Polish patients. Also the survivals of Polish women with carcinoma of the uterine cervix are significantly lower as compared with other countries.

[Histological types of lung cancer in urban and rural inhabitants of the Krakow region]. / Typy histologiczne raka pluca wsród ludnosci miejskiej i wiejskiej regionu krakowskiego.

Incidence of different histological types of lung cancer was analyzed in the years 1978-1984 in city and rural inhibitants of the Cracov region. Histologically verified lung cancer ranged from 21% in females of rural population to 52% of city male inhabitants. Squamous lung cancer followed by small cell lung cancer were most often diagnosed in city population and males from villages. Adenocarcinoma was seen more often in females, being most often diagnosed in females of rural population.

Trends and patterns in lung cancer incidence in four cities of Eastern Europe, 1971-1980.

The trends of age-adjusted and age-specific lung cancer incidence rates showed over the period of 1971-1980 substantial differences in four selected towns of Eastern Europe. The downward age-adjusted trends in males coincided in Berlin with the possible beginning of their downturn in the whole country, while their decline in both sexes in Bratislava could be related to the change of demographis factors. In the other two towns studied--Cracow and Tallinn--the substantial increase of lung cancer incidence in males corresponded with the similar evolution in both respective countries (Poland and Estonia). The rising trends in females in both mentioned towns could be more or less compared with their dramatic increase in some developed countries. The rising trends of age-specific rates in younger age groups of males do not indicate meanwhile the positive influence of low-tar or filter-tipped cigarettes on lung cancer incidence in males observed recently in some countries.