The impact of an expanded genetic testing program and selective oophorectomy on the incidence of ovarian cancer in West Pomerania.

The aim of the study was to evaluate the impact of a regional population-based genetic testing program on the incidence of ovarian cancer in West Pomerania. Between 1999 and 2010, a total of 37,552 women ages 35 to 70 were tested for three BRCA1 founder mutations at the outpatient genetics clinic of the Pomeranian Medical University in Szczecin, Poland. A total of 641 women were found to carry a mutation (1.7%) and of these, 220 had a prophylactic oophorectomy (34.3%). A total of 12 women had an occult cancer diagnosed at the time of prophylactic oophorectomy (5.5%). We estimate that 26 more ovarian cancers would have been diagnosed by January 2015 in the absence of these oophorectomies and that an additional 25 cancers will be prevented in the future (total 51). During this period, 1611 ovarian cancers were diagnosed in the region; therefore we estimate that approximately 1.6% of ovarian cancers were prevented between 1999 and 2015 by our genetic testing program. We conclude that the prophylactic oophorectomies performed between 1999 and 2010 as a result of widespread BRCA1 mutation testing have reduced the incidence of ovarian cancer in Pomerania by a small amount (about 1.6%), and that the impact of genetic testing will increase in the coming years.

Concordance of KRAS mutation status between luminal and peripheral regions of primary colorectal cancer. A laser-capture microdissection-based study.

The presence of KRAS mutation in colorectal cancer (CRC) is a marker of resistance to anti-EGFR therapy. However, there are conflicting reports concerning intratumoral heterogeneity of KRAS mutations. The aim of this study was to determine whether within primary CRCs with KRAS mutations intratumoral KRAS mutation heterogeneity can be detected between two strictly defined areas, i.e. the luminal (mucosa/submucosa) and peripheral invasive front of the tumor. Using laser-capture microdissection, from every tumor about 400-500 nests of cancer cells were excised from each of the examined areas (luminal and peripheral) and PNAClamp, a high-sensitivity real-time PCR-based diagnostic assay for KRAS mutation testing, was used for molecular analysis. KRAS mutations were detected in codon 12 in both luminal and peripheral regions in all tumors examined. We conclude that from the point of view of practical KRAS mutation testing for predictive purposes in patients with CRC (i.e. testing mutations in codons 12 and 13) sampling errors are unlikely to occur if in CRCs with KRAS mutations only the luminal (as in biopsy tissue) or peripheral region is examined, provided a sensitive system of detection is applied and an appropriate number of tumor cells with minimal contamination by benign cells is analyzed.

Immunohistochemical analysis of steroid receptors in ovaries of postmenopausal women--effects of aging and hormone status.

Current knowledge on immunolocalization and immunoexpression of steroid hormone receptors, especially estrogen receptor alpha (ER-alpha), progesterone receptor (PR) and androgen receptor (AR) in normal ovaries in postmenopausal women is not complete. The recognition of localization of these receptors in postmenopausal women is crucial, as many of these women receive estro-progestagene therapy, and its participation in the pathogenesis of ovarian cancer should be carefully studied. In our paper we present the results of immunohistochemical studies performed on samples from 100 post-menopausal women (aged: 48 to 60 years) who did not use hormonal therapy. The ovaries were removed during elective operation due to uterine leiomyoma, endometriosis and/or prolapsed uterine. PR, ER-alpha and AR were detected in the normal ovaries of postmenopausal women in stroma and in ovarian surface epithelium, as well as in its invagination and in epithelial inclusion cysts. The expression of PR and AR did not change, while the expression of ER-alpha decreased in time from menopause, and it was also detected in patients more than 10 years after menopause. Women older than 60 were not included in the study. The concentration of selected hormones was measured in the serum. The immunohistochemical expression of PR and AR were similar in all examined patients and did not correlate with FSH, LH, T, A, DHEAS concentrations in serum, while immunohistochemical expression of ER-alpha correlated with FSH, LH, T, A, DHEAS concentrations in serum. The significant correlation of decreasing expression of ER-alpha in normal ovarian tissue and decreasing concentrations of T, A and DHEAS in serum were found, as well as increasing serum concentrations of FSH and LH.

BRCA1-positive breast cancers in young women from Poland.

We identified 4316 unselected incident cases of early-onset breast cancers (<51 ears of age at diagnosis) in 18 Polish hospitals between 1996 and 2003. We were able to obtain a blood sample for DNA analysis from 3472 of these (80.4%). All cases were tested for the presence of three founder mutations in BRCA1. The proportion of cases with a BRCA1 mutation was 5.7%. The hereditary proportions were higher than this for women with breast cancer diagnosed before age 40 (9%), for women with cancer of medullary or atypical medullary histology (28%), for those with bilateral cancer (29%) or with a family history of breast or ovarian cancer (13%). It is reasonable to offer genetic testing to women with early-onset breast cancer in Poland.

p21/WAF1/Cip1 expression in invasive ductal breast carcinoma: relationship to p53, proliferation rate, and survival at 5 years.

The p21/WAF1/Cipl antibody, DCS-60, was characterized by means of immunoblotting and immunofluorescence on a variety of human breast cancer cell lines. Heterogeneous staining of nuclei was observed with strong staining of cells in early G1. p21/WAF1/Cipl expression in invasive ductal, not otherwise specified breast carcinomas was determined using immunohistochemistry with this antibody and computerized image analysis. Two hundred and twenty-two tumors, including 130 from patients with no axillary node involvement, were examined. p21-positive tumor cell nuclei were found in 30% of the breast carcinomas. The percentage of tumor cell nuclei that were positive ranged from less than 1% to greater than 10%. In the whole cohort of patients, p21 expression was significantly associated with a low histological grade. In the node-negative group, there was a significant negative correlation between p21 positivity and a high (>10%) MIB-1 score. The mean MIB-1 score was significantly lower in p21-positive tumors in the whole cohort of patients (P=0.03) and in the nodenegative group (P=0.02). No association was found between p21 expression and overall survival at 5 years. With respect to p21/p53 phenotype, the significant difference in survival was noted only for the group of patients treated with adjuvant chemotherapy. The p21- p53+ phenotype had the worst survival (58% surviving 5 years), while the p21+ p53- phenotype had good survival (83% surviving 5 years; P<0.05). The results seem to suggest a correlation between p21/p53 phenotype and response to adjuvant chemotherapy.

Quality control in cytodiagnosis of carcinoma of the cervix.

In many European countries the morbidity and mortality due to cancer of the cervix are too high. Even in countries, where smears have been taken at regular intervals for many years, the percentage of new cases does not decrease as much as it was expected. There are several reasons that may explain this situation. At least two of them can be solved by pathologists: quality control (QC) system and organization of a school of gynaecological cytopathology for pathologists and cytotechnicians. Here we focus on the main aspects of External and Internal QC and new approaches to QC, including new methods and stricter control of the professional level of all persons involved in the screening and diagnosis of cervical cancer.

Value of pedigree/clinical data, immunohistochemistry and microsatellite instability analyses in reducing the cost of determining hMLH1 and hMSH2 gene mutations in patients with colorectal cancer.

The aim of this study was to evaluate the significance of pedigree/clinical data, immunohistochemistry (IHC) and microsatellite instability (MI) analyses in the reduction of costs of constitutional hMLH1 and hMSH2 gene mutation diagnosis in patients with colorectal cancers (CRC). Pedigree/clinical data were evaluated on a series of 168 patients with CRC, including 43 consecutive sporadic late-onset and 25 consecutive, definitive or suspected hereditary non-polyposis colorectal cancer (HNPCC) cases, examined by IHC and MI analyses. In the latter group, 6/25 (24%) constitutional mutations were found. We detected no germline mutations in the sporadic late-onset patients. The lowest costs (880 Euro/mutation detected) were achieved by performing pedigree/clinical data (for exclusion of late-onset sporadic CRC) in conjuction with IHC only. In this model 1/6 (17%) mutations was missed. Additional preselection by IHC and MI analyses before sequencing was required to detect all mutations. In this approach, which seems to be the most effective in the search for hMLH1 and hMSH2 gene mutation, the cost was 1767 euro/mutation detected.

Clinico-pathological characteristics of colorectal cancer and serum anti-p53 antibodies.

The purpose of this study was to correlate the presence of p53 antibodies in sera of patients with colorectal adenocarcinoma with size, site and stage of the tumour, age and sex of a patient and the level of carcinoembryonic antigen (CEA) in the serum. p53 antibodies were detected using enzyme-linked immunoabsorbent assay (ELISA). Serum p53 antibodies were detected in 30 of 145 patients (21%), mostly in Astler-Coller stage B1 (28% of patients). No association was found between p53 antibody status in stage A+B1+B2 vs stages C1+C2+D (22% vs 19%) i.e. between patients without and with metastases to regional lymph nodes and/or distant metastases. Serum p53 antibodies were detected in 9 of 34 patients (26%) with tumour localised in the right part vs 21 of 109 patients (19%) with tumours in the left part of the colon and in 18 of 96 (19%) of patients with tumours localised in rectosigmoideum vs 12 of 47 (26%) with tumours in the remaining colon. There was no significant correlation between serum anti p53 antibody and CEA statuses. Increased level of serum CEA was seen in 46/145 (32%) patients. Patients with C1+C2+D stage cancers had high serum CEA level more frequently than did patients with A+B1+B2 stage tumours (44% vs 19% respectively, p < 0.001). Of 102 cases with normal CEA level, 19 (19%) were positive for anti p53 antibodies. These results together with the literature data [11, 20] indicate that approximately 27% CEA negative patients may have serum p53 antibodies. Therefore simultaneous assessment of serum p53 antibodies and CEA seems to be useful for monitoring high risk patients and for postoperative patient monitoring.

Nuclear morphometry of MIB-1 positive and negative tumor cells in primary and metastatic malignant melanoma of the skin.

The purpose of this study was the evaluation of nuclear area, nuclear axis ratio, perimeter and roundness of nuclei of tumor cells with and without Ki-67 antigen expression (demonstrated immunohistochemically using MIB-1 antibody) in primary and metastatic malignant melanoma of the skin. The parameters were further analyzed with respect to their association with the depth of malignant invasion according to Clark [7] and tumor thickness according to Breslow [6]. 142 malignant melanomas (53 primary and 89 metastatic), were assessed employing a computerized image analyzer Quantimet 600S (Leica). The mean nuclear area of MIB-1 positive nuclei was significantly larger than that of the negative ones (p < 0.0001) both in primary and metastatic malignant melanoma. In comparison to the primary melanoma the nuclei of metastatic melanoma cells had a larger area and were more rounded, while the MIB-1 positive nuclei additionally showed a greater degree of polymorphism of their area and shape. With growing invasion thickness according to Breslow and increased Clark's level, the mean nuclear area of tumor cells increased, and their shape became more round. The MIB-1 positive tumor cell nuclei of primary melanomas with metastases were significantly out of round in comparison to primary melanomas without metastases. The results indicate an association between the area and shape of melanoma cell nuclei and the presence of metastases, and between the nuclear area of tumor cells and such factors related to poor prognosis as the depth of invasion and the tumor thickness.

Absence or reduction of Fhit expression in most clear cell renal carcinomas.

The FHIT gene at human chromosome region 3p14.2 straddles the common fragile site, FRA3B, and numerous homozygous deletions in cancer cell lines and primary tumors. Also, the 3p14.2 chromosome breakpoint of the familial clear cell kidney carcinoma-associated translocation, t(3;8)(p14.2;q24), disrupts one FHIT allele between exons 3 and 4, fulfilling one criterion for a familial tumor suppressor gene: that one allele is constitutionally inactivated. Because the FHIT gene sustains biallelic intragenic deletions rather than mutations, there has not been evidence that the FHIT gene frequently plays a role in kidney cancer, although replacement of Fhit expression in a Fhit-negative renal carcinoma cell line suppressed tumor growth in nude mice. We have now assessed 41 clear cell renal carcinomas for expression of Fhit by immunohistochemistry. Normal renal tubule epithelial cells express Fhit uniformly and strongly, whereas 51% of the tumors are completely negative, 34% of tumors show a mixture of positive and negative cells, and 14% are uniformly positive, although usually less strongly positive than the normal epithelial cells. Most interestingly, there was a correlation between complete absence of Fhit and the G1 morphological grade and early clinical stage. Morphological grades G2 and G3 exhibited a mixture of positive and negative cells with a tendency for a higher fraction of negative cells in G3. Fhit inactivation is likely to be an early event in G1 tumors and may be associated with progression in G2 and G3 tumors.

The clinical significance of p53 accumulation in gastric carcinoma.

BACKGROUND: Alterations in the expression of p53 tumor suppressor protein is a frequent event in human cancer but the practical implications of this phenomenon are yet to be fully exploited. The objective of this study was to determine the value of p53 accumulation as a marker of tumor progression and prognosis of gastric carcinoma patients and to evaluate whether this parameter can be properly assessed prior to surgery. METHODS: The expression of p53 was studied immunohistochemically in 200 gastric carcinomas using paraffin embedded surgical specimens and endoscopic biopsies. The correlation between p53 expression in tumor tissue, selected clinicopathologic variables, and the course of the patients' disease were analyzed. RESULTS: Results showed that 42.5% of the gastric carcinomas expressed elevated levels of p53 protein. P53 accumulation positivity correlated with increasing tumor stage and size (P < 0.001 and P = 0.025, respectively). P53 positive tumors had a higher propensity for lymph node and distant metastases (P < 0.001). P53 accumulation was also more frequently detected in carcinoma from proximal rather than distal stomach (P = 0.027). In patients receiving potentially curative resection for advanced cancer, p53 accumulation was an independent parameter and the strongest for poor prognosis (RR = 3.7, P < 0.001). There was complete concordance between immunohistochemical detection of p53 in endoscopic and surgical material. CONCLUSIONS: A preoperative assessment of p53 expression in gastric carcinoma can be helpful to identify patients at high risk of metastatic spread to regional lymph nodes and independently to identify those with especially poor prognosis. When combined with routine procedures, this simple and inexpensive test might allow appropriate planning of better treatment strategies.

Proliferation of cancer cells and stromal cells in colorectal adenocarcinomas as assessed with anti-PCNA/cyclin monoclonal antibody.

Monoclonal anti-proliferating cell nuclear antigen antibody (PCNA, PC 10) was used to measure proliferation of tumor cells and stromal cells in 27 colorectal adenocarcinomas. Mean proliferation index of cancer cells was 33.7% (SD +/- 16.3) and that of stromal cells 14.3 (SD +/- 12.5). Positive correlation between proliferation index of cancer cells and proliferation rate of stromal cells was found (r = 0.64, p < 0.001). Correlation between proliferation of stromal lymphocyte-like cells and fibroblast-like cells was noticed (r = 0.69, p < 0.001).

Proliferation cell nuclear antigen (PCNA) expression in cervical intraepithelial neoplasia (CIN). An immunohistochemical study.

We analyzed the expression of proliferating cell nuclear antigen (PCNA) in ectocervical sections fixed in formalin and embedded in paraffin using monoclonal antibody PC10, PAP and APAAP methods. A total of 156 cases was studied: 32 cases with normal epithelium, 33 cases with CIN I, 36 cases with CIN II, 55 cases with CIN III. We evaluated: 1. the index of PCNA (IPCNA), i.e. the proportion of PCNA-positive cells relative to all cells in the epithelium; 2. the IPCNA in each epithelial layer; 3. the epithelial height with PCNA-positive cells relative to the total height of the epithelium; 4. changes in the IPCNA with respect to the presence or absence of histological signs of HPV infection. We found: 1. a significant correlation between the index of PCNA and the severity of dysplasia in the whole epithelium and its individual layers; 2. a significant correlation between the epithelial height with PCNA-positive cells relative to the total height of the epithelium and the severity of dysplasia; 3. a lack of correlation between the IPCNA and HPV(+) and HPV(-) cases.

Proliferating cell nuclear antigen (PCNA) expression in laryngeal squamous cell carcinomas. An immunohistochemical study.

The relationship between proliferating cell nuclear antigen (PCNA) expression and clinical stage, lymph node status, histological grade, malignancy index (MI) [13] were studied in 103 laryngeal squamous cell carcinomas (SCC). The PCNA index (the percentage of PCNA positive nuclei) was examined immunohistochemically using monoclonal PC10 antibody on paraffin sections. PCNA immunoreactivity was seen in all samples with mean value of PCNA index 37.4% for supraglottic and 36.8% for glottic SCC. The PCNA index was significantly related to histological grade and malignancy index (p < 0.05). For all tumors no correlation was found between PCNA index and clinical stage and lymph node status however, metastasizing glottic SCC had higher PCNA index than non-metastasizing ones. The modification of MI in glottic laryngeal SCC by adding PCNA index as a new parameter is proposed.

Immunohistochemical assessment of proliferation rate of breast carcinoma cells using Ki-67, MIB-1 and anti-PCNA monoclonal antibodies.

MIB-1 Ki-67 and PCNA scores in infiltrating ductal NOS breast carcinomas were compared. The correlation between MIB-1, Ki-67 and PCNA indices and several clinicopathological factors that have prognostic significance in breast cancer was also assessed. The mean Ki-67, MIB-1 and PCNA indices were 13.4%, 19.4%, 27.6%, respectively. Significant positive linear correlation was found only between Ki-67 and MIB-1 indices. PCNA score did not correlate with Ki-67 and MIB-1 indices. The significant correlation between Ki-67 and MIB-1 scores and histological grade was found. There was no correlation between Ki-67 and MIB-1 indices and axillary lymph node status or tumor diameter. The results suggest that MIB-1 antibody is an excellent tool for assessment of proliferative rate of breast cancer cells in paraffin sections.

Prognostic significance of tumor cell proliferation rate as determined by the MIB-1 antibody in breast carcinoma: its relationship with vimentin and p53 protein.

The prognostic value of tumor cell proliferative activity as measured by the MIB-1 monoclonal antibody in invasive ductal not otherwise specified breast carcinomas was determined for 186 patients, including 111 with no axillary node involvement. The MIB-1 antibody detects the Ki-67 antigen in microwave-processed paraffin sections of the formalin-fixed tumors. The mean MIB-1 score was 16% for all tumors, 16% for the node-negative group, and 15% for the node-positive group. In univariate survival analysis, the MIB-1 score (dichotomized, /=10%) predicted overall 5-year survival in all of these groups. The mean MIB-1 score was significantly higher in vimentin- and p53 protein-positive tumors (P > 0.001) than in negative ones. The impact of vimentin expression and of p53 positivity on the prognostic significance of the tumor cell proliferation rate was assessed. Vimentin was associated significantly with poor 5-year survival in the entire cohort, and a particularly strong association was found in the node-negative group. p53 had a weak but statistically nonsignificant influence on survival. In a multivariate analysis using the Cox proportional hazards model, vimentin (P = 0.0002) was the only independent prognostic factor in node-negative patients. In contrast, the MIB-1 score (P = 0.009) was the only independent prognostic factor in the node-positive group. Therefore, node-negative patients with vimentin-positive tumors and node-positive patients with tumors with high proliferation rates might be appropriate candidates for early adjuvant chemotherapy.

Elevated cathepsin D expression in kainate-evoked rat brain neurodegeneration.

Expression patterns of cathepsin D (lysosomal aspartic protease) and glial fibrillary acidic protein (GFAP, a marker of reactive astroglia) were determined by Northern blot analysis and immunohistochemistry in the rat brain during neurodegeneration accompanying kainate-evoked seizures. The level of cathepsin D mRNA in the hippocampus, limbic cortex, and temporo-parieto-occipital neocortex was shown to increase, starting at 6 h after kainate treatment, and reaching peak values at 3-7 days after the neurotoxin administration. A similar time course of elevated accumulation was noted for GFAP mRNA in these structures. Immunohistochemical analysis performed 3 days after kainate treatment showed that the increased cathepsin D levels were confined mainly to the degenerating neurons in the susceptible brain areas, while the elevated GFAP immunoreactivity was observed in reactive astrocytes. Although cathepsin D and GFAP expression levels were elevated by kainate administration, their expression patterns revealed significant differences with regard to both intensity and site of induction.

Cathepsin D in invasive ductal NOS, medullary, lobular and mucinous breast carcinoma. An immunohistochemical study.

Cathepsin D expression was determined by immunohistochemistry in 445 formalin fixed, paraffin embedded primary invasive breast carcinomas. We found: 1. a relationship between cathepsin D expression and histological type of tumour, 2. a negative correlation between cathepsin D expression and histological grade, 3. a negative correlation between cathepsin D expression and tumour diameter, 4. a relationship between the morphology of cathepsin D granules and histological type of the tumour.

Immunohistochemical analysis of bcl-2 and p53 expression in breast carcinomas: their correlation with Ki-67 growth fraction.

We examined 59 breast cancers for p53 and bcl-2 protein expression by immunohistochemistry. The results were correlated with Ki-67 immunostaining. p53-negativity was noted in 40 cases and the remaining 19 tumours were p53-positive. Thirty-six tumours showed strong expression of bcl-2 and in 23 no staining for this protein was observed. We found statistically significant reverse correlation between expression of p53 and bcl-2 in majority of carcinomas: 31 cases were bcl-2 positive and p53-negative, and 14 tumours were bcl-2-negative and p53-positive. Six carcinomas showed no nuclear staining for Ki-67 and in the remaining 53 the percent of cancer cells positive for Ki-67 ranged from 1 to 60 (mean: 14.6). In these 53 cases we found that bcl-2-positive tumours were characterized by lower proliferation than bcl-2-negative tumours, the mean value of Ki-67 immunostaining being 10.7% and 23.0%, respectively. p53-negative tumours showed lower proliferation than p53-positive tumours: mean Ki-67 index was 10.2% and 23.9%, respectively. We conclude that immunohistochemically detected p53 and bcl-2 proteins show a significant inverse relationship in majority of breast carcinomas and their expression correlates with tumour proliferation (Ki-67 immunostaining).