MiR-221/-222 differentiate prognostic groups in advanced breast cancers and influence cell invasion.

BACKGROUND: MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells. METHODS: MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT-PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222. RESULTS: In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed. CONCLUSION: This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.

Impact of genomic stability on protein expression in endometrioid endometrial cancer.

BACKGROUND: Genomic stability is one of the crucial prognostic factors for patients with endometrioid endometrial cancer (EEC). The impact of genomic stability on the tumour tissue proteome of EEC is not yet well established. METHODS: Tissue lysates of EEC, squamous cervical cancer (SCC), normal endometrium and squamous cervical epithelium were subjected to two-dimensional (2D) gel electrophoresis and identification of proteins by MALDI TOF MS. Expression of selected proteins was analysed in independent samples by immunohistochemistry. RESULTS: Diploid and aneuploid genomically unstable EEC displayed similar patterns of protein expression. This was in contrast to diploid stable EEC, which displayed a protein expression profile similar to normal endometrium. Approximately 10% of the differentially expressed proteins in EEC were specific for this type of cancer with differential expression of other proteins observed in other types of malignancy (e.g., SCC). Selected proteins differentially expressed in 2D gels of EEC were further analysed in an EEC precursor lesion, that is, atypical hyperplasia of endometrium, and showed increased expression of CLIC1, EIF4A1 and PRDX6 and decreased expression of ENO1, ANXA4, EMD and Ku70. CONCLUSION: Protein expression in diploid and aneuploid genomically unstable EEC is different from the expression profile of proteins in diploid genomically stable EEC. We showed that changes in expression of proteins typical for EEC could already be detected in precursor lesions, that is, atypical hyperplasia of endometrium, highlighting their clinical potential for improving early diagnostics of EEC.

Calculating dissolved marine oxygen values based on an enhanced Benthic Foraminifera Oxygen Index.

Marine oxygen minimum zones (OMZs) trap greenhouse gases, reduce livable habitats, a critical factor for these changes is the amount of dissolved oxygen (DO). The frequently used tool to reconstruct DO values, the Benthic Foraminifera Oxygen Index (BFOI), showed major shortcomings and lacks effectiveness. Therefore, we enhanced the BFOI and introduce enhanced BFOI (EBFOI) formulas by using all available data benthic foraminifers provide, calculating the whole livable habitat of benthic foraminifers, including bottom water oxygenation (BWO) and pore water oxygenation (PWO). Further, we introduce for the first time a transfer function to convert EBFOI vales directly into DO values, increasing efficiency by up to 38%. All formulas are calibrated on modern samples and applied to fossil datasets. Our new approach provides a major improvement in defining and reconstructing marine oxygen levels and eutrophication, by, providing a new toolset for understanding past changes and tracking actual and predicted future expanding OMZs.

Differential expression of ANXA6, HSP27, PRDX2, NCF2, and TPM4 during uterine cervix carcinogenesis: diagnostic and prognostic value.

BACKGROUND: Cytology-based diagnostics of squamous cervical cancer (SCC) precursor lesions is subjective and can be improved by objective markers. METHODS: IHC-based analysis of ANXA6, HSP27, peroxiredoxin 2 (PRDX2), NCF2, and tropomyosin 4 (TPM4) during SCC carcinogenesis. RESULTS: Expression of ANXA6, HSP27, PRDX2, and NCF2 in the cytoplasm of dysplastic cells increased from cervical intraepithelial neoplasia 2/3 (CIN2/3) to microinvasive cancer. Invasive SCC showed lower expression of TPM4 than CIN and normal epithelium. CIN2/3 with the highest sensitivity and specificity differed from normal epithelium by cytoplasmic expression of HSP27. Patients with cytoplasmic HSP27 expression in SCC deviating from that observed in normal epithelium had worse relapse-free (P=0.019) and overall (P=0.014) survival. Invasive SCC with the highest sensitivity and specificity differed from normal epithelium by expression of PRDX2 and TPM4 in the cytoplasm, from CIN2/3 by the expression of ANXA6 and TPM4 in the cytoplasm, and from microinvasive SCC by the expression of PRDX2 and ANXA6 in the cytoplasm. The number of sporadic ANXA6+ cells between the atypical cells increased from CIN2/3 to invasive SCC. CONCLUSION: Detection of expression changes of the proteins ANXA6, HSP27, PRDX2, NCF2, and TPM4 in SCC precursor lesions may aid current cytological and pathological diagnostics and evaluation of prognosis.

Prevention of tumour cell dissemination in diagnostic needle procedures.

BACKGROUND: A side effect of diagnostic needle biopsies is the possibility to disseminate tumour cells into the needle track, which may cause concern in certain malignant tumour types. METHODS: In order to prevent tumour cell dissemination we developed a technology that uses radiofrequency (RF) pulses to sterilise the needle track and denaturate tumour cells. To determine feasibility, we applied this technology to fine needle aspiration biopsy (FNAB) and used breast cancer as a model tumour. Routine FNAB was performed in 88 patients with adenocarcinoma and blood droplets passing the skin orifice were cytomorphologically analysed for the presence of tumour cells. RESULTS: The analysis showed the presence of tumour cells in 65/88 cases (74%). When using an experimental anti-seeding device in a subset of patients viable tumour cells were found in 0/31 cases (P<0.001). In all 31 patients blood passing the skin orifice was sparse. No degrading effect on the cytological sample inside the needle was detected and pain caused by the RF pulses was comparable to that of the biopsy procedure itself. CONCLUSION: The herein presented method has the potential to prevent the dissemination of viable tumour cells in the needle track and minimize bleeding without additional pain or degradation of the aspirate.

In situ quantification of HER2-protein tyrosine kinase 6 (PTK6) protein-protein complexes in paraffin sections from breast cancer tissues.

BACKGROUND: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. METHOD AND RESULTS: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6-HER2 protein-protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). CONCLUSION: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2-PTK6 complexes are of prognostic relevance.

Differential tissue-specific protein markers of vaginal carcinoma.

The objective was to identify proteins differentially expressed in vaginal cancer to elucidate relevant cancer-related proteins. A total of 16 fresh-frozen tissue biopsies, consisting of 5 biopsies from normal vaginal epithelium, 6 from primary vaginal carcinomas and 5 from primary cervical carcinomas, were analysed using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry. Of the 43 proteins identified with significant alterations in protein expression between non-tumourous and tumourous tissue, 26 were upregulated and 17 were downregulated. Some were similarly altered in vaginal and cervical carcinoma, including cytoskeletal proteins, tumour suppressor proteins, oncoproteins implicated in apoptosis and proteins in the ubiquitin-proteasome pathway. Three proteins were uniquely altered in vaginal carcinoma (DDX48, erbB3-binding protein and biliverdin reductase) and five in cervical carcinoma (peroxiredoxin 2, annexin A2, sarcomeric tropomyosin kappa, human ribonuclease inhibitor and prolyl-4-hydrolase beta). The identified proteins imply involvement of multiple different cellular pathways in the carcinogenesis of vaginal carcinoma. Similar protein alterations were found between vaginal and cervical carcinoma suggesting common tumourigenesis. However, the expression level of some of these proteins markedly differs among the three tissue specimens indicating that they might be useful molecular markers.

Time-sensitive reversal of hyperplasia in transgenic mice expressing SV40 T antigen.

The role of viral oncoprotein expression in the maintenance of cellular transformation was examined as a function of time through controlled expression of simian virus 40 T antigen (TAg). Expression of TAg in the submandibular gland of transgenic mice from the time of birth induced cellular transformation and extensive ductal hyperplasia by 4 months of age. The hyperplasia was reversed when TAg expression was silenced for 3 weeks. When TAg expression was silenced after 7 months, however, the hyperplasia persisted even though TAg was absent. Although the polyploidy of ductal cells could be reversed at 4 months of age, cells at 7 months of age remained polyploid even in the absence of TAg. These results support a model of time-dependent multistep tumorigenesis, in which virally transformed cells eventually lose their dependence on the viral oncoprotein for maintenance of the transformed state.

A new specially designed needle significantly increases sample yield during fine needle aspiration of breast lesions.

A large and increasing number of women in the western world will at some point during their life be investigated morphologically for breast cancer. Fine needle aspiration (FNA) is one morphological method which is considered to be the fastest, cheapest and the most patient-friendly approach. Nevertheless, the technique has not gained major clinical success outside of Scandinavia, mainly because of a high frequency of insufficient samples. With this in mind it is quite peculiar that standard needles which are mainly configured for blood sampling and infusion therapy, comprising large quantities of residual spaces, are used. In this study we have developed and tested a new needle dedicated for FNA, which is intended to abate this drawback by increasing the sampling yield by changing the tip angle, the cannula wall-thickness and the storage compartment. In total, 499 consecutive aspiration procedures of palpable breast lesions were performed to compare the new needle (outer diameter 0.6mm) with standard needles (outer diameters 0.6 mm and 0.7 mm). The new needle provided three times more material than did standard needles of the same diameter. Surprisingly, the new needle also provided more material than the standard 0.7 mm needle, which increased up to almost twice the material in cases with no material in the syringe. The frequency of tests with sparse harvested material (< 4 mg) was less with the new needle (9%) compared to its standard counterpart (35%). The presented results were obtained by a very skilled sampling operator. Thus for the average sampling operator who probably obtains more samples in the spare range, the new dedicated FNA needle should have even more added value.

A new semi-automated instrument to improve the fine needle aspiration procedure during breast lesion cell sampling.

A large and increasing number of women in the western world will at some point during their life be investigated morphologically for some type of breast lesion. Fine Needle Aspiration (FNA) is one morphological method which is considered to be the fastest, cheapest and the most patient-friendly approach. However, the frequency of conclusive samples using this method varies and is often too low, especially when performed by unexperienced operators. In this study we have developed and tested a new semi-automated instrument ("CytoTest") designed for FNA which is intended to improve the efficacy of the technique by increasing the percentage of conclusive samples. A total of 443 consecutive aspiration procedures on palpable breast lesions were performed to compare this new "CytoTest" equipment with the standard protocol using the same type of needles. We conclude that by increasing the extent and frequency of the reciprocatory motions used by an experienced sampling operator as well as enhancing the ejection pressure, the cellular yield can be increased almost three folded compared to the standard protocol. For cases with high amounts of non-diagnostic material (such as blood or cystic fluid) which were discarded, up to four times more sample could be obtained. Furthermore, the frequency of sparse samples under 1mg was halved with use of the "CytoTest".

Genomic instability and proliferative activity as risk factors for distant metastases in breast cancer.

The role of genomic instability and proliferative activity for development of distant metastases in breast cancer was analysed, and the relative contribution of these two risk factors was quantified. A detailed quantitative comparison was performed between Ki67 and cyclin A as proliferative markers. The frequency of Ki67 and cyclin A-positive cells was scored in the same microscopic areas in 428 breast tumours. The frequency of Ki67-positive cells was found to be highly correlated with the frequency of cyclin A-positive cells, and both proliferation markers were equally good to predict risk of distant metastases. The relative contribution of degree of aneuploidy and proliferative activity as risk markers for developing distant metastases was studied independently. Although increased proliferative activity in general was associated with an increased risk of developing distant metastases, ploidy level was found to be an independent and even stronger marker when considering the group of small (T1) node negative tumours. By combining proliferative activity and ploidy level, a large group of low risk breast tumours (39%) could be identified in which only a few percentage of the tumours (5%) developed distant metastases during the 9-year follow-up time period.

Ki-67 expression predicts locoregional recurrence in stage I oral tongue carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) is an aggressive cancer associated with poor prognosis. Methods for determining the aggressiveness of OTSCC from analysis of the primary tumour specimen are thus highly desirable. We investigated whether genomic instability and proliferative activity (by means of Ki-67 activity) could be of clinical use for prediction of locoregional recurrence in 76 pretreatment OTSCC paraffin samples (stage I, n=22; stage II, n=33; stage III, n=8; stage IV, n=13). Eleven surgical tumour specimens were also analysed for remnants of proliferative activity after preoperative radiotherapy. Ninety-seven percent of cases (n=72) were characterised as being aneuploid as measured by means of image cytometry. Preoperative radiotherapy (50-68 Gy) resulted in significant reduction of proliferative activity in all patients for which post-treatment biopsies were available (P-value=0.001). Proliferative activity was not associated with response to radiation in stage II patients. However, we report a significant correlation between high proliferation rates and locoregional recurrences in stage I OTSCC patients (P-value=0.028). High-proliferative activity is thus related to an elevated risk of recurrence after surgery alone. We therefore conclude that Ki-67 expression level is a potentially useful clinical marker for predicting recurrence in surgically treated stage I OTSCC.

Prognostic value of protein tyrosine kinase 6 (PTK6) for long-term survival of breast cancer patients.

The cytoplasmic tyrosine kinase PTK6 (BRK) shows elevated expression in approximately two-thirds of primary breast tumours, and is implicated in EGF receptor-dependent signalling and epithelial tumorigenesis. Using immunohistochemistry, we performed a retrospective study on 426 archival breast cancer samples from patients with long-term follow-up and compared the protein expression levels of PTK6, the HER receptors, Sam68 (a substrate of PTK6), and signalling proteins including MAP kinase (MAPK), phosphorylated MAPK (P-MAPK), and PTEN. We show that PTK6 expression is of significant prognostic value in the outcome of breast carcinomas. In multivariate analysis, the disease-free survival of patients of >or=240 months was directly associated with the protein expression level of PTK6 (P

Laminin-5 as a marker of invasiveness in cervical lesions.

BACKGROUND: Treatment decisions for cervical cancer, a common disease worldwide, depend on demonstrating whether or not tumor invasion of the surrounding tissue has occurred. Invasion can be difficult to assess by standard histopathologic methods, especially when limited amounts of tissue are available. Several studies of a variety of cancers have reported increased expression of laminin-5-an important attachment protein for epithelial cells-in invasive carcinomas. This study was designed to investigate whether the presence of laminin-5 is related to the invasive capacity of cervical lesions. METHODS: We used immunohistochemical methods to stain archival, paraffin-embedded sections of cervical lesions with a polyclonal antibody specifically targeting the gamma2 chain of human laminin-5 protein. The study sample included 23 lesions of mild and moderate dysplasia (cervical intraepithelial neoplasia [CIN] 1 and 2, respectively), 32 lesions of severe dysplasia or carcinoma in situ (CIN 3), 15 lesions of microinvasive cancer, and 20 lesions of frankly invasive cancer. Cellular proliferative activity was also investigated by the use of monoclonal MIB-1 (directed against the antigen Ki-67) and anticyclin A antibodies. RESULTS: Invasiveness of cervical lesions was positively associated with immunohistochemical staining of the gamma2 chain of laminin-5 (two-sided P =.001). All CIN 1 and CIN 2 lesions-except one CIN 2 lesion later shown to be invasive cancer-and 21 CIN 3 lesions tested negative for the gamma2 chain of laminin-5. Eleven CIN 3 lesions and all invasive cancers tested positive for this protein. One lymph node metastasis and a pleural metastasis from one of the patients with invasive cancer showed strong immunohistochemical positivity. Proliferative activity increased with advancement of the lesion but was not confined to cells positive for the gamma2 chain of laminin-5. CONCLUSIONS: These data suggest that antibodies directed against the gamma2 chain of laminin-5 can identify cervical lesions with invasive capacity and thus may be useful as a sensitive marker of early invasion.

Prognostic significance of nuclear DNA content in mammary adenocarcinomas in humans.

The value of the determination of the DNA content of tumor cells for the assessment of the prognosis of mammary adenocarcinoma was studied in 36 patients, who survived for at least 15 years after the cancer had been found and in another 42 patients who died within a 2-year period of diagnosis. The results show a distinct correlation between the type of the DNA histograms of the carcinoma and the grade of cancer. This correlation was particularly apparent among patients surviving for at least 15 years despite the occurrence of metastases or relapses after primary treatment, i.e., in patients not cured by their first treatment.

Reversibility of bronchial cell atypia.

Various degrees of cellular atypia were induced in the bronchial epithelium of dogs by means of repeated submucous 20-methylcholanthrene injections. Thereafter, the 20-methylcholanthrene treatment was stopped, and the outcome of the bronchial cell atypias in individual dogs was studied using cytomorphological and cytochemical methods. The results suggest that the various degrees of 20-methylcholanthrene-induced cellular atypias, including those cytologically interpreted as malignant, may reflect reversible cellular alterations which disappear after removal of the carcinogen. Similar observations were made in a group of cigarette smokers who, after malignant-appearing cells were observed in the sputum material, stopped smoking or significantly reduced their cigarette consumption.

Prognostic significance of nuclear DNA content in serous ovarian tumors.

Specimens from 73 serous ovarian cancers were examined with respect to DNA content of the tumor cells. The prognostic value of DNA analysis, as reflected in patient survival, was retrospectively compared with that of conventional histological assessment of cancer. DNA in individual tumor cells was measured in sections from the original paraffin-embedded specimens. High proportions of cells with very high DNA values were identified in tumors from 16 patients, 10 of whom died of the disease during the follow-up period. The histological classification was invasive adenocarcinoma in 47 tumors and borderline cancer in 26. All of the patients who died had invasive adenocarcinoma. Although both DNA analysis and histological evaluation were sensitive predictors of mortality, the specificity of the DNA method was distinctly higher (0.90 versus 0.42). The study suggests that analysis of the DNA content of tumor cells can be a useful supplement to histological assessment of cancer and accordingly can significantly assist in the planning of treatment.

p53 deletion as a genetic marker in urothelial tumor by fluorescence in situ hybridization.

In order to investigate the significance of p53 deletion, 42 specimens of transitional cell carcinoma were analyzed by interphase cytogenetics with a fluorescence in situ hybridization technique and compared with clinicopathological and cytochemical parameters. In total, 27 (64%) and 16 (38%) specimens demonstrated p53 deletion and overexpression, respectively. The p53 deletion was significantly correlated with grade (P < 0.01), stage (P < 0.05), S-phase fraction (P < 0.05), and DNA ploidy (P < 0.01), while p53 overexpression correlated only with grade (P < 0.05). The close correlation of p53 deletion with clinicopathological parameters suggests p53 deletion to be of clinical importance to indicate the malignant potential of human urothelial tumors.

Comparison of nuclear DNA content in primary and metastatic papillary thyroid carcinoma.

Nuclear DNA values were determined in 40 primary papillary thyroid carcinomas, as well as in 52 corresponding local recurrences and metastases were observed either at the time of diagnosis or up to 20 years later. The patient population consisted of 34 survivors and 6 nonsurvivors. In survivors, both the primary tumors and their recurrences and metastases exhibited a majority of cells with DNA values within the normal diploid region, whereas nonsurvivors showed increased and scattered DNA values. In all cases, the primary tumors and the corresponding recurrences and metastases showed similar DNA distribution patterns even if many years had passed between the detection of the primary tumor and the metastases. The results indicate that in papillary thyroid carcinomas, the DNA distribution patterns in the primary tumor and the corresponding recurrences or metastases are generally similar throughout the entire period of disease.

Genetic aberrations in adrenocortical tumors detected using comparative genomic hybridization correlate with tumor size and malignancy.

The differentiation between malignant and benign adrenocortical tumors is often difficult, and better markers are required. Because the genetic background of adrenocortical tumors is poorly characterized, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes in 8 sporadic primary adrenocortical cancers and 14 adenomas. There was a strong relationship between the number of genetic aberrations detected using CGH and both tumor size and malignancy. No alterations were seen in the smaller adenomas (< 5 cm), whereas the two largest adenomas (5 cm each) and seven of the eight cancers (7-20 cm) showed an increased number of genetic alterations. The presence of genetic aberrations detected using CGH was associated with an aneuploid DNA pattern. In the cancers, losses most often involved the chromosomal regions 2, 11q, and 17p (four of eight tumors), whereas gains took place at chromosomes 4 and 5 (four of eight tumors). In conclusion, our data indicate that genetic changes may help to define the malignant potential of adrenocortical tumors. Furthermore, the CGH results implicate several chromosomal regions that may contain genes with an important role in the development of adrenocortical cancers.