Marek Cybulski

Immunohistochemical Analysis of MIB-1 Proliferative Activity in Human Endometrial Cancer. Correlation with Clinicopathological Parameters, Patient Outcome, Retinoblastoma Immunoreactivity and K-Ras Codon 12 Point Mutations

To test the prognostic utility of MIB-1 in human endometrial neoplasias, the proliferative activities of fifty-two endometrial carcinomas obtained from Polish women were assessed. We also investigated the relationship between the MIB-1 Proliferative Index and the well-known clinicopathological features of cancer (clinical stage, histological type, histological grade, depth of myometrial invasion), patients age, overall survival, retinoblastoma immunostaining and K-ras codon 12 point mutations. The mean MIB-1 Proliferation Index was 43.8%, with a median of 36.0%. Due to the great intratumour heterogeneity of the immunoreaction, the Index ranged from 0% to 98%. A significant relationship was noted between MIB-1 expression and histological grading (p=0.0004) and myometrial invasion of cancer (p=0.01). Multivariate Cox regression demonstrated that the clinical stage was the only independent prognostic factor during follow-up (p=0.025). There was a tendency towards a poorer outcome for women with a Proliferative Index of >31% than for patients whose Index was ≤ 31%; the difference, however, did not reach significance (p=0.25; log-rank test). Interestingly, uterine cancers lacking retinoblastoma protein expression had a mean MIB-1 Proliferation Index that was nearly twice as high as in those neoplasias that stained positively for retinoblastoma (70.33% and 42.14%, respectively; p=0.09; Mann-Whitney-U test). There were no significant differences between K-ras codon 12 point mutation-positive and -negative endometrial carcinomas regarding the proliferative activity of the cancer (mean Indexes 47.6% and 43.8%, respectively; p=0.66, Mann-Whitney-U test). Our data support the view that MIB-1 proliferative activity was significantly increased with a decrease of the histological grading and with the myometrial invasion of human endometrial cancer.

Sialic acid concentration in serum and tissue of endometrial cancer patients.

Serum total sialic acid (TSA) level was determined in the group of 45 patients with endometrial neoplasia. Moreover, the sialic acid content in 23 surgically obtained tumor tissue specimens has been estimated. The mean value of serum total sialic acid level of the cancer patients (2.38 mmol/l) was significantly higher (P < 0.0001) than the sialic acid level in the control group (1.52 mmol/l). The elevation of serum total sialic acid level was associated with the burden of the tumor (2.30 mmol/l and 2.68 mmol/l for stages I and II + III, respectively). Tissue sialic acid content showed no significant differences between early and advanced clinical stage of the disease.

Allelic Loss at TP53 Is Not Related to p53 Protein Overexpression in Primary Human Endometrial Carcinomas

We examined loss of heterozygosity (LOH) at the TP53 gene in primary human endometrial carcinomas (EC), and investigated the relationship between allelic loss, p53 protein overexpression, pRb-1 pathway alterations and MIB-1 proliferative activity. Applying the non-isotopic PCR-RFLP/VNTR-silver staining techniques, we investigated TP53 LOH in 46 tumors at four polymorphic loci. Out of 42 informative carcinomas, LOH was found in 19% of the cases studied. In general, there was no significant relationship between LOH and the clinical and pathological variables of cancer, including patient age, clinical stage, histological grade or depth of myometrial invasion. Interestingly, none of 7 tumors associated with hyperplasia revealed allelic imbalance, whereas 8 of 27 (30%) tumors without hyperplasia exhibited LOH (p = 0.312; Fisher’s exact test). Overexpression of nuclear p53 was not correlated with allelic loss at TP53 (p = 0.336, Fisher’s exact test). It is worth pointing out that p53 immunoreactivity was significantly related to proliferative activity of cancer (R = 0.42, p = 0.0037; Spearman’s rank correlation test). A tendency towards a poorer outcome was reported in EC patients displaying TP53 LOH during short-time follow-up (p = 0.093; log-rank test). None of the tumors simultaneously showed LOH at TP53 and RB1 genes(R = –0.211, p = 0.16; Spearman’s rank correlation test). p16INK4A alterations (LOH and gene deletion) occurred concomitantly, with 3 tumors showing the TP53 allelic loss, whereas the cyclin D1/cdk4 complex was overexpressed in a case with TP53 LOH. Altogether, losses at TP53 were not associated with p53 nuclear overexpression, but may affect a subset of EC patients characterized by an unfavorable prognosis at short-time follow-up. Allelic loss at TP53 seems to arise independently of LOH at the RB1 gene in carcinomas of the uterine corpus in humans. Disruptions at p16INK4A and/or cdk4/cyclin D1 concomitantly occurring with TP53 LOH may participate in the development of a subset of endometrioid-type ECs.

The implementation of an organised cervical screening programme in Poland: an analysis of the adherence to European guidelines

BackgroundWell-organised quality-controlled screening can substantially reduce the burden of cervical cancer (CC). European guidelines (EuG) for quality assurance in CC screening provide guidance on all aspects of an organised screening programme. Organised CC screening in Poland was introduced in 2007. The purpose of our study was to analyse: (i) adherence of the programme to EuG; (ii) programme process and performance indicators; (iii) impact of the programme on the incidence of and mortality from CC.MethodsAvailable data on the policy, structure and functioning of the Polish programme were compared with the major points of the EuG. Data on the process, and available performance indicators were drawn from the screening database and other National Health Fund (NHF) systems. Joinpoint regression was used to assess changes in CC incidence and mortality trends.ResultsThe Polish programme adheres partially to EuG in terms of policy and organisation. Only a limited set of performance indicators can be calculated due to screening database incompleteness or lack of linkage between existing databases. The screening database does not include opportunistic smears collected within NHF-reimbursed or private care. The organised programme coverage rate fluctuated from 21% to 27% between 2007-2013. In 2012 the coverage reached 35% after combining both organised and opportunistic smears reimbursed by the NHF. In 2012 the number of smears reimbursed by NHF was 60% higher in opportunistic than in organised screening with significant overlap. Data from the private sector are not recorded. Depending on years, 30-50% of women referred for colposcopy/biopsy because of abnormal Pap smears were managed within the programme. The age-standardised CC incidence and mortality dropped linearly between 1999 and 2011 without evidence of a period effect.ConclusionsThe Polish organised cervical screening programme is only partially adherent to evidence-based EuG. Its implementation has not influenced the burden of CC in the country so far. Changes with special focus on increasing coverage, development of information systems and assessment of quality are required to increase programme adherence to EuG and to measure its effectiveness. Our findings may be useful to improve the Polish programme and those implemented or planned in other countries.

Cervical Cancer Histology, Staging and Survival before and after Implementation of Organised Cervical Screening Programme in Poland

A population-based organised cervical cancer screening programme (OCCSP) was introduced in Poland in 2006. In this study we have aimed to analyse whether selected parameters related to invasive cervical cancer (ICC) of patients diagnosed in two distant gynaecological oncology centres changed after the first screening round of the programme run between 2006–2008. We have run a retrospective cross-sectional analysis of 189 women diagnosed with ICC between 2002–2005 (directly before introduction of the programme) and 165 patients diagnosed between 2009–2012 (just after the first screening round of the programme) and compared their age at diagnosis, histology, stage of tumours and overall survival (OS). Mean age of patients diagnosed in years 2002–2005 and 2009–2012 was 52.1 and 52.6 years respectively. Squamous cell carcinomas constituted 90.5% and 86.1% of tumours diagnosed in years 2002–2005 and 2009–2012 respectively and the rest of tumours had glandular and other histologies. 74.5% and 61.0% of women diagnosed in years 2002–2005 and 2009–2012 respectively had early ICC (FIGO—International Federation of Gynaecology and Obstetrics stages I-IIA) and the rest had advanced disease (FIGO IIB-IV). We have noticed no significant differences in mean age of patients, histology of tumours and OS of patients with ICC diagnosed before and after the first screening round of OCSSP in Poland. Advanced stages of ICC were more commonly diagnosed after the introduction of OCSSP. Changes only in some clinical parameters of patients with ICC were noticed before and after the first screening round of OCSSP in Poland but OS of patients remained the same.

Prognostic value of paraoxonase 1 in patients undergoing coronary artery bypass grafting surgery

Background The aim of this study was to evaluate whether –108C/T polymorphism of the paraoxonase 1 (PON1) gene and the plasma enzyme activity are risk factors for adverse cardiac events after coronary artery bypass grafting (CABG). Material/Methods Seventy-one patients with coronary heart disease (CHD) undergoing CABG were enrolled in the study. Genomic DNA was extracted from the venous blood using the Gen Elute™ Blood Genomic DNA kit (Sigma) according to the manufacturer’s instructions. PON1 activity was measured in 50 mM glycine/NaOH buffer (pH 10.5) containing 1.0 mM paraoxon, and 1.0mM CaCl2. Results The mean PON1 activity toward paraoxon and toward phenyl acetate was equal (166.5±86.9 U/ml and 96.0±47.2 U/ml, respectively) in patients with CHD. The –108C/T polymorphism of PON1 gene was tested. In CABG patients, PON1 activities in dependence on genotypes were significantly different and equalled 266.2±117.9 U/ml for CC, 178.8±64.7 U/ml for CT, and 98.9±59.2 U/ml for TT genotype. Patients with PON1 activity lower than 193.5 U/ml exhibited significantly increased risk of a serious cardiac event in comparison with patients with PON1 activity higher or equal to this value (p=0.03). Additionally, TT genotype was significantly associated with shorter time of event-free survival in comparison with CT and CC genotypes (p=0.009). Conclusions The PON1 polymorphism and enzyme plasma activity are associated with CHD occurrence. High PON1 activity connected with the presence of CC and CT genotypes decreases the recurrence of symptoms of coronary heart disease and improve prognosis after CABG.

Molecular bases of aberrant miR-182 expression in ovarian cancer.

The molecular bases of miR‐182 deregulation in epithelial ovarian cancers (EOCs) remain unknown and its diagnostic or prognostic role in EOCs is still unclear. We performed miR‐182 expression analysis using a microarray approach and real‐time PCR (qPCR). We also used array comparative genomic hybridization and methylated DNA immunoprecipitation to study copy number changes and methylation aberrations within coding locus/promoter sequences of miR‐182 in EOC tissues, respectively. We have found that miR‐182 expression is significantly increased in EOC (P < 0.00001) and that higher miR‐182 expression in EOC is linked with significantly shorter overall survival (P = 0.026). The methylation of miR‐182 promoter was significantly associated with lower miR‐182 expression in EOC tissues (P = 0.045). miR‐182 over‐expression is connected with copy number (CN) gains of this miRNA coding sequences in EOC (P = 0.002), and the number of PRDM5 copies is significantly and inversely correlated with miR‐182 expression evaluated by qPCR (R = −0.615, P = 0.009). We conclude that the aberrant miR‐182 expression in EOC may be due to CN gains within its coding locus. The miR‐182 promoter is rarely methylated in EOC, and its methylation status is associated with lower miR‐182 expression. Deletion of the PRDM5 locus may play a supportive role in miR‐182 overexpression in EOC. miR‐182 is an unfavorable prognostic factor in EOC.

Sialosyl-Tn expression in normal and pathological conditions of human endometrium. An immunohistochemical study

To assess the clinico-prognostic relevance of the cell surface carbohydrate glycoprotein in normal and pathological conditions of human endometrium, Sialosyl-Tn (STn) antigen was immunohistochemically studied in normal (n = 10), hyperplastic (n = 18), and neoplastic (n = 60) endometrial lesions. There was no STn antigen reactivity in the proliferative endometrial slides, while weak staining was observed in all secretory endometria. STn expression was noted in 8/18 (44%) hyperplastic endometrial cases and in 40/60 (67%) endometrial carcinomas. Positive staining was observed throughout the cytoplasm of the glandular cancer cells, at the cell membranes, and in an intraluminar mucus. This antigen was mostly expressed heterogeneously as far as the distribution of positive cells is concerned. There was a statistically significant association between STn expression and the histological grading of cancer (p = 0.019). Advanced clinical stage (III-IV; p = 0.014) and infiltration of the myometrial wall (more than 1/2 of the myometrial wall; p = 0.004), but no STn immunoreactivity, were reported to be independent prognostic variables during follow-up. Our study shows that a) STn is not constantly expressed during the menstrual cycle, and is increased at the secretory phase of the cycle; b) Sialosyl-Tn reactivity decreases with the degree of tumor differentiation, but there was no relationship with other clinicopathological variables of cancer; c) this cell surface carbohydrate glycoprotein does not appear to predict the outcome of endometrial cancer patients.

Reimbursed Costs of Management of Uterine Cervical Lesions in Poland--a Descriptive Analysis of Data from the National Health Fund and the Ministry of Health.

BACKGROUND Despite implementation of organised screening programme in 2006/2007, cervical cancer (CC) incidence and mortality in Poland are still higher than the average in the European Union. CC and preceding cervical intraepithelial neoplasia (CIN) caused by human papillomaviruses (HPVs) can be prevented by vaccines which are reimbursed in around 20 European countries but not in Poland. CC and CIN can be also detected with the use of HPV tests which are not included in the Polish screening programme. Reimbursement for HPV vaccines and HPV testing requires cost-effectiveness analyses which include country-specific data on the burden and costs of management of cervical neoplasia. Therefore, we investigated the burden of cervical neoplasia and direct costs associated with its detection and management in Poland in 2012 reimbursed by the National Health Fund (NHF) - the only public healthcare insurance institution. We also report administrative costs of the organised screening programme covered by the Ministry of Health. METHODS Data on the burden and reimbursed costs of organised and opportunistic screening as well as management of cervical neoplasia were drawn from the NHF databases. Numbers of women reported with CIN and CC were ascertained. RESULTS In 2012, there were 765,266 and 1,288,358 reimbursed Pap smears collected within and outside the organised screening programme, respectively. Expenditures on medical and administrative procedures in organised screening reached PLN (Polish Zloty) 41,470,664 and 12,150,398 respectively. The number of women with particular diagnosis and reimbursement for the management of these lesions were as follows: glandular ectropion 208,033 and PLN 37,349,515; CIN1 10,521 and PLN 6,616,375; CIN2 5,812 and PLN 5,071,155; CIN3 6,487 and PLN 7,611,062; unspecified grade CIN 36,575 and PLN 12,352,034; and CC 33,482 and PLN 52,377,006, respectively. In women with ectropion and CIN the total number of local excision/ablative therapeutic procedures on the cervix reached 47,658 and the total number of hysterectomies was 1,321. CONCLUSION In 2012, management of approximately 93 thousand women with HPV-related cervical lesions reimbursed in Poland amounted to PLN 84,027,632 which makes it a considerable public health problem. The number of women managed for glandular ectropion is considerable and related costs are high. Total reimbursement for detection, treatment and follow-up of all cervical lesions reaches at least PLN 137 million annually.

The pattern of p14ARF expression in primary and metastatic human endometrial carcinomas: correlation with clinicopathological features and TP53 pathway alterations.

Objectives: Alterations of p53 pathway (p14ARF/MDM2/p53) play a crucial role in the development and progression of various human neoplasms, including endometrial carcinoma (EC). The aim of the current research was to examine the p14ARF expression pattern in primary ECs and corresponding metastatic lesions. Materials and Methods: We studied 47 primary ECs and corresponding metastatic lesions applying immunohistochemistry and investigated the relationship between p14ARF overexpression and clinicopathological variables of carcinoma as well as TP53 alterations. Results: Protein expression was predominantly nuclear, present in 32 (68%) of 47 primary cases and in 28 (60%) of 47 metastatic lesions. There were seven p14ARF-positive primary tumors showing negative reactivity in the metastatic lesions. On the other hand, 3 cases lacked protein immunoreactivity in the primary ECs but revealed weak nuclear staining in the corresponding metastases. A case of primary cervical adenocarcinoma metastasizing to the lymph nodes showed p14ARF expression both in the primary tumor and the corresponding metastases. A trend was found between the p14ARF expression in primary tumors and the presence of the neoplasms in the fallopian tube (P = 0.063), but none of the other clinicopathological variables of carcinoma was related to protein immunoreactivity in advanced-stage uterine neoplasms. The p14ARF expression in EC metastases was related to the presence of the primary tumor in the fallopian tube (P = 0.036). The p14ARF expression was not associated with unfavorable outcome both in the primary tumors (P = 0.302) and in the corresponding metastases (P = 0.217). There was also no relationship between the p14ARF expression pattern and TP53 pathway alterations. Conclusions: Altogether, the p14ARF protein is expressed in more than half of the primary ECs and metastatic lesions analyzed and is associated with the transtubal dissemination of the primary tumor. The pattern of the p14ARF expression is not associated with the alterations of other TP53 pathway members in advanced-stage human ECs.