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.

Distribution of T-lymphocytes and B-lymphocytes in peripheral blood and effusions of patients with cancer.

The relative distribution of T- and B-lymphocytes in the blood and in pleural or abdominal effusions was compared among 24 patients with fluid accumulation due to metastatic cancer and 8 patients without evidence of cancer. The data obtained indicated that the mean percentage of T-lymphocytes in malignant effusions was significantly greater than that in the peripheral blood of the same patients. At the same time, the mean eprcentage of B-lymphocytes was decreased in malignant effusions when compared with peripheral blood. Neither of these differences was observed when effusions and blood of patients with nonmalignant effusions were compared. In addition, patients with both types of effusions had fewer total lymphocytes in their blood than did normal control patients, whereas those with cancer-associated effusions had an increased proportion of active T-lymphocytes in their blood.

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.

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.

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.

p53 protein and vimentin in invasive ductal NOS breast carcinoma--relationship with survival and sites of metastases.

p53 protein and vimentin status were available from immunocytochemical studies of 253 formalin-fixed paraffin-embedded invasive ductal not otherwise specified (NOS) carcinomas from patients for whom follow-up data was also on file. For the 127 node-negative patients, multivariate analysis showed a highly significant correlation between p53 and vimentin (P < 0.001), a strong correlation between vimentin and probability of survival to 90 months but only a weak association between p53 and survival to 90 months. p53 also never entered trees of prognostic indicators derived using stepwise regression with Kaplan-Meier statistics for node-negative and node-positive subgroups, while vimentin status dominated the node-negative trunk. In addition, p53 and vimentin status were analysed versus the site of the first distant metastasis for node-negative and node-positive patients. Analysis by p53 status showed no significant effect on visceral metastases. In contrast, vimentin-positive primaries metastasised twice (and in node-negative patients, 3.5 times) as often to lung, liver and brain as did the vimentin-negative primaries. Both p53-positive and vimentin-positive tumours showed a significantly lower tendency to metastasise to the bone than did their negative counterparts.

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).

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.

A 19-year follow-up of multiple juvenile hyaline fibromatosis.

Nineteen years observation of two siblings with multiple juvenile hyaline fibromatosis is presented. This entity was described 14 years ago. It is concluded that this disease can be effectively controlled by surgical excision of all newly discovered subcutaneous tumors.

Transmission and scanning electron microscopic studies of cells in effusions.

The transmission (TEM) and scanning electron microscopic (SEM) features of cancer cells and benign cells from human effusions were described. Both in the TEM and the SEM the following cell types were indentified; mesothelial cells, histiocytes, lumphocytes, eosinophilic leukocytes, neutrophilic leukocytes, plasma cells, erthrocytes and cancer cells. The ultrastructure of malignant cells originating from cancers most commonly metastisizing to the serous body cavities was described in detail. Both the TEM and the SEM clearly demonstrated fundamental differences between the surfaces of the cancer cells and benign cells in body cavity fluids: cancer cells were covered by numerous microvilli, while non-neoplastic cells were not. The approach described in this paper can be used for purposes of cytologic diagnosis in difficult preselected cases and may also have some important theoretical implications.

Configuration of surfaces of human cancer cells obtained by fine needle aspiration biopsy: a comparative light microscopic and scanning electron microscopic study.

The surface configuration of human cancer cells removed from their natural setting by fine needle aspiration biopsies was studied by scanning electron microscopy. Cells from several carcinomas of the breast, malignant melanomas, oat cell carcinomas and a malignant lymphoma were the subject of this study. Only viable cells, identified by light microscopy prior to scanning, were selected for study. Contrary to prior observations on cancer cells in effusions, the surface configuration of human cancer cells obtained directly from tissues is variable and may depend significantly on the histologic patterns.

Differential diagnosis of lymph node aspirates by intermediate filament typing of tumor cells.

Twenty-one lymph node aspirates for which a differential diagnosis was difficult or not possible by light microscopy alone were selected for further study by intermediate filament typing. The use of four monoclonal antibodies specific for different keratin subsets or for vimentin resulted in a definitive diagnosis in all instances. The results show that use of these antibodies can improve the accuracy of cytologic diagnosis and provide further evidence that intermediate filament typing can help differentiate (1) malignant melanoma from adenocarcinoma, (2) malignant lymphoma from small-cell anaplastic carcinoma and (3) squamous-cell carcinoma from adenocarcinoma. Intermediate filament typing can also be used to identify very small numbers of carcinoma cells in specimens that are apparently negative for tumor cells by light microscopy. The method is quick, relatively simple, reliable and unambiguous, provided that appropriate questions are asked and antibodies with well-defined specificities are used.

Diagnostic significance of coexpression of intermediate filaments in fine needle aspirates of human tumors.

A study was undertaken of the diagnostic significance of the coexpression of intermediate filaments in fine needle aspirates of human tumors. Three types of coexpression were found: (1) true coexpression, in which tumor cells simultaneously express more than one intermediate filament protein; (2) pseudocoexpression, in which various tumor cell types from histogenetically different parts of a complex tumor show different results; and (3) false coexpression, in which tumor cells with one or two types of intermediate filaments are present together with benign cells expressing a different filament type. True coexpression of vimentin and keratin was documented in renal cell carcinomas, endometrial carcinomas, certain thyroid carcinomas and Hürthle cell adenomas. Coexpression of keratin and neurofilaments was seen in Merkel cell carcinomas, and coexpression of desmin and vimentin was found in leiomyosarcomas. Keratin, vimentin and neurofilament expression was seen in medullary thyroid carcinomas, and keratin, vimentin and glial fibrillary acidic protein expression was observed in pleomorphic adenomas of the salivary gland. Pseudocoexpression was noted in synovial sarcoma, epithelioid sarcoma, benign cystosarcoma phyllodes of the breast, teratocarcinoma, malignant granular cell tumor, progonoma, Wilms' tumor and triton tumor. Sources of false coexpression are also discussed.

T and B lymphocyte enumeration in the diagnosis of lymphocyte-rich pleural fluids.

The presence of a monotonous population of small lymphocytes in effusions may be due to chronic lymphatic leukemia (CLL), malignant lymphoma of the small cell type (SLML) or a chronic inflammatory process, such as tuberculosis. In this report we provide evidence that the enumeration of T and B lymphocytes in such fluids may provide a reliable means of distinguishing between malignant and nonmalignant effusions. The mean percentage of T lymphocytes in th pleural effusions of ten patients wit nonmalignant diseases was 80.2, and the mean percentage of B lymphocytes was 7.4. In marked contrast, B cells predominated (mean, 83.3%) in the pleural effusions of patients with lymphoma or leukemia. Since the great majority of chronic lymphocytic lymphomas and leukemias are of B-cell origin, we conclude that effusions containing a large predominance of B cells have a high probability of being malignant. Those that contain a predominance of T cells are almost always benign, although one should always consider the rare occurrence of chronic lymphocytic lymphomas and leukemias of T-cell origin.

Fine needle aspiration cytology of malignant melanoma of the skin.

Fifty malignant melanomas of the skin were aspirated with a fine needle, and the cytomorphologic features of the smears were evaluated. An attempt was made to establish the frequency of morphologic features found in melanoma cells. The following features were regarded as frequent: lack of cohesiveness between the malignant cells; binucleated, multinucleated and giant cells; and intranuclear "vacuoles." The nuclei usually had a regular outline, and the nuclear chromatin was evenly distributed. The majority of the smears contained a mixture of epithelial-type and spindle cells. The nuclei of the epithelial-type cells were usually in an eccentric position. The morphologic features of malignant cells in smears, as described, can be helpful in making the preoperative cytologic diagnosis of nonpigmented malignant melanoma of the skin.

Intermediate filament typing of tumor cells in fine needle aspirates by means of monoclonal antibodies.

Well-characterized monoclonal antibodies directed against different intermediate filament proteins were used in the typing of tumor cells in 30 fine needle aspiration (FNA) biopsy specimens received for routine cytologic examination to assess the value of intermediate filament typing in FNA cytology. Tumors from sites that included liver, large bowel, pancreas, breast, skin, thyroid, thigh and kidney were examined with monoclonal antibodies specific for either all keratins or keratin subsets, vimentin, desmin or specific neurofilament polypeptides. Intermediate filament typing helped to confirm, revise or refine the diagnoses made by light microscopy and provided information of value in the classification of tumors of uncertain origin.

Cytodiagnosis of acinic cell carcinoma. Ultrastructural study of material obtained by fine needle aspiration biopsy.

The material obtained by fine needle aspiration biopsy from a metastatic acinic cell carcinoma was evaluated by light and electron microscopy. A preliminary diagnosis of acinic cell carcinoma established by light microscopy was confirmed by electron microscopic study. Glandular arrangement of cancerous cells and the presence of a specific type of secretory granules in their cytoplasm were the most characteristic ultrastructural features of the acinic cell carcinoma.

Fine needle aspiration cytology of intraocular malignant melanoma.

The techniques used for fine needle aspiration biopsy of 19 intraocular tumors clinically suspected to be malignant melanomas are described. Aspirates of 16 tumors yielded adequate material for the cytologic diagnosis of malignant melanoma. Malignant cells of the epithelial type and of a spindle shape were found in the smears. The main cytologic features of the epithelial-type cells were eccentrically located nuclei, distinct nucleoli, binucleation and multinucleation. The spindle-shaped cells, dispersed or arranged in clusters, were characterized by centrally placed, oval or rodlike nuclei, with or without nucleoli. Three types of smears were distinguished: (1) those with a predominance of epithelial-type cells, (2) those with a predominance of spindle-shaped cells and (3) those with mixed epithelial-type and spindle-shaped cells. Our observations prove that precise morphologic diagnosis of intraocular malignant melanoma and its cellular types can be established by fine needle aspiration biopsy. However, we do not advocate fine needle aspiration biopsy as a routine diagnostic procedure in all intraocular melanomas. This method should be reserved for only cases that cannot be definitely diagnosed by conventional techniques or in which the cellular type of melanoma must be defined in order to choose the proper method of treatment.

The ultrastructure of surfaces of positively identified cells in the human urinary sediment. A correlative light and scanning electron microscopic study.

Surfaces of cells in the urinary sediment, postively identified by light microscopy (LM) were studied by scanning electron microscopy (SEM). About 35% of benign urothelial cells were characterized by the presence of short, stubby microvilli (MV). About 60% of such cells had surface microridges, and the remaining cells had surface blebs. Squamous epithelial cells were characterized primarily by microridges or by a mixture of microridges with short microvilli. Urothelial cancer cells had various surface characteristics. About 30% of these cells displayed plemorphic MV, about 10% of these cells showed short, regular MV, and the remainder appeared to be poorly preserved and had smooth surfaces, either with MV remnants or points of membrane rupture (holes or pits). The correlation between SEM surface features and the LM appearance of urothelial cancer cells was poor. Many of the most characteristic cancer cells on LM had no distinguishing SEM features. It is concluded that the application of SEM for the diagnosis of urothelial carcinoma in urine is of uncertain benefit, pending further studies.