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Tweetable abstract It is well accepted that pharmacogenomics (PGx) information from Asia and Europe should not be applied to Africa. More work is needed on different ethnic groups to generate population-specific algorithms that can be used effectively and safely.

The continuous emergence of new SARS-CoV-2 variants required rapid and reliable diagnostic methods for early detection and monitoring of the spread of the virus, especially in low-resource countries where whole genome sequencing is not available. We aimed to evaluate and compare the performance of two different RT-qPCR screening assays for the detection of B.1.617 lineage mutations. A total of 85 SARS-CoV-2 positive samples were collected between 9

The issue of school reopening has raised several concerns; therefore, the parent's opinion is essential to consider. This study aimed to evaluate the parent's attitudes and concerns toward school reopening in the COVID-19 era. A cross-sectional survey was performed using in-person self-administered questionnaires, the data was collected in the period between January and April 2021 covering parents' concerns and attitudes toward school reopening. A total of 402 parents participated in the survey. Analysis showed that 56.7% of parents have agreed with school reopening, but 54% have raised some legitimate concerns. Importantly, there was a strong correlation between parents' opinions towards school reopening and their level of education, and their concerns about their children's safety if the school was reopened. Despite parents' concerns, it does seem that slightly over half were in favour of school reopening and would send their children to school only if the schools did apply strict precautions and restriction measures. Sharing parents' views toward school reopening with school leaders and decision-makers is important to assess the feasibility and effectiveness of return to schools and to improve existing prevention programs.

Alpha (B.1.1.7) SARS-COV-2 variant was detected in September 2020 in minks and humans in Denmark and UK. This variant has several mutations in the spike region (S) which could increase the transmissibility of the virus 43-90% over previously circulating variants. The National Center for Disease Control (NCDC) announced on 24th February 2021 a 25% frequency of B.1.1.7 strain in Libya using a reverse-transcriptase quantitative PCR assay. This assay relies on the specific identification of the H69-V70 deletion in S gene which causes its failure of amplification (SGTF). This deletion is not specific for B.1.1.7; but is also characteristic of two other SARS-COV-2 variants. This study aimed to estimate the frequency of B.1.1.7 and identify other variants circulating in Libya in February 2021. We performed whole genome sequencing of 67 positive SARS-COV-2 samples collected on 25th February 2021 in Libya which were also tested by RT-qPCR for SGTF. Our results showed that 55% of samples had mutations specific to B.1.525 strain and only ∼3% of samples belonged to B.1.1.7. These findings suggested that B.1.525 was spreading widely in Libya. The use of such RT-qPCR assay although useful to track some variants, it cannot discriminate between variants with H69-V70 deletion. RT-qPCR assays could be multiplexed to identify multiple variants and screen samples prior to sequencing. We emphasize on the need for providing whole-genome sequencing to the main COVID-19 diagnostic laboratories in Libya as well as establishing international collaboration for building capacity and advancing research in this time of the pandemic.

There is a great demand for more rapid tests for SARS‐CoV‐2 detection to reduce waiting time, boost public health strategies for combating disease, decrease costs, and prevent overwhelming laboratory capacities. This study was conducted to assess the performance of 10 lateral flow device viral antigen immunoassays for the detection of SARS‐CoV‐2 in nasopharyngeal swab specimens. We analyzed 231 nasopharyngeal samples collected from October 2020 to December 2020, from suspected COVID‐19 cases and contacts of positive cases at Biotechnology Research Center laboratories, Tripoli, Libya. The performance of 10 COVID‐19 Antigen (Ag) rapid test devices for the detection of SARS‐CoV‐2 antigen was compared to a quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR). In this study, 161 cases had symptoms consistent with COVID‐19. The mean duration from symptom onset was 6.6 ± 4.3 days. The median cycle threshold (C t) of positive samples was 25. Among the 108 positive samples detected by RT‐qPCR, the COVID‐19 antigen (Ag) tests detected 83 cases correctly. All rapid Ag test devices used in this study showed 100% specificity. While tests from six manufacturers had an overall sensitivity range from 75% to 100%, the remaining four tests had a sensitivity of 50%–71.43%. Sensitivity during the first 6 days of symptoms and in samples with high viral loads (C t, Highlights Rapid antigen testing has high sensitivity and specificity in early disease when patients present less than 7 days of symptom onsetPatients should be encouraged to test within one week after getting COVID‐19 related symptoms and within 24 hrs. if they develop disturbed smell/taste.The use of rapid antigen testing is important for reducing burden on molecular diagnostic laboratories.

Restriction of mobility between countries is an important regulatory measure to combat pandemics such as the coronavirus disease 2019 (COVID-19). Currently, PCR testing is required to enter the Libyan borders. However, no post-travel quarantine is employed. In this report, we briefly discuss travel regulations in Libya during the COVID-19 pandemic and disease importation by travelers. The results showed that almost half of the sample travel because of health care and therapy reasons. Tunisia was the most visited destination mainly for trading and business and receiving healthcare. Importantly, 13% of asymptomatic travelers were SARS-CoV-2 positive. Issues regarding repeated testing among very frequent travelers and variant importation needs to be addressed in a more efficient manner.

Lamins A/C, a major component of the nuclear lamina, play key roles in maintaining nuclear integrity, regulation of gene expression, cell proliferation and apoptosis. Reduced lamin A/C expression in cancer has been reported to be a sign of poor prognosis. However, its clinical significance in breast cancer remains to be defined. This study aimed to evaluate expression and prognostic significance of lamin A/C in early-stage breast cancer. Using immunohistochemical staining of tissue microarrays, expression of lamin A/C was evaluated in a large well-characterised series of early-stage operable breast cancer (n = 938) obtained from Nottingham Primary Breast Carcinoma Series. Association of lamin A/C expression with clinicopathological parameters and outcome was evaluated. Positive expression rate of lamin A/C in breast cancer was 42.2% (n = 398). Reduced/loss of expression of lamin A/C was significantly associated with high histological grade (p

Preoperative (chemo)radiotherapy, (C)RT, is an essential part of the treatment of rectal cancer patients, but tumor response to this therapy among patients is variable. Thus far, there are no clinical biomarkers that could be used to predict response to (C)RT or to stratify patients into different preoperative treatment groups according to their prognosis. Overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) has been demonstrated in several cancers and is frequently associated with reduced survival. Recently, high CIP2A expression has also been indicated to contribute to radioresistance in head and neck squamous cell carcinoma, but few studies have examined the connection between CIP2A and radiation response regarding other malignancies. We have evaluated CIP2A protein expression levels in relation to tumor regression after preoperative (C)RT and survival of rectal adenocarcinoma patients. The effects of CIP2A knockdown by siRNA on cell survival were further investigated in colorectal cancer cells exposed to radiation. Patients with low‐CIP2A‐expressing tumors had more frequently moderate or excellent response to long‐course (C)RT than patients with high‐CIP2A‐expressing tumors. They also had higher 36‐month disease‐specific survival (DSS) rate in categorical analysis. In the multivariate analysis, low CIP2A expression level remained as an independent predictive factor for increased DSS. Suppression of CIP2A transcription by siRNA was found to sensitize colorectal cancer cells to irradiation and decrease their survival in vitro. In conclusion, these results suggest that by contributing to radiosensitivity of cancer cells, low CIP2A protein expression level associates with a favorable response to long‐course (C)RT in rectal cancer patients.

Cancer is one of the leading causes of death worldwide, despite the large efforts to improve the understanding of cancer biology and development of treatments. The attempts to improve cancer treatment are limited by the complexity of the local milieu in which cancer cells exist. The tumor microenvironment (TME) consists of a diverse population of tumor cells and stromal cells with immune constituents, microvasculature, extracellular matrix components, and gradients of oxygen, nutrients, and growth factors. The TME is not recapitulated in traditional models used in cancer investigation, limiting the translation of preliminary findings to clinical practice. Advances in 3D cell culture, tissue engineering, and microfluidics have led to the development of "cancer-on-a-chip" platforms that expand the ability to model the TME in vitro and allow for high-throughput analysis. The advances in the development of cancer-on-a-chip platforms, implications for drug development, challenges to leveraging this technology for improved cancer treatment, and future integration with artificial intelligence for improved predictive drug screening models are discussed.

Aim: To assess the predictive and prognostic value of neurofibromin (NF) expression in colorectal carcinoma (CRC). Materials and Methods: The present series consists of archival samples from 191 patients with stage I, II, III, or IV CRC treated between 1981 and 1990 at the Turku University Hospital (Finland). Tumor biopsies as microarray blocks were analyzed for expression of NF by immunohistochemistry. Different grading systems were tested for NF expression. Results: A significant correlation between NF expression and tumor localization was found, with tumors arising in the colon showing intense NF expression more often than those arising in the rectum (p=0.014). Higher expression of NF was more common in tumors not responding to treatment (p=0.004). Tumors with multiple metastases showed higher expression of NF than those with single metastasis only (p=0.025). Furthermore, NF expression showed a borderline (p=0.068) correlation with gender; tumors of women showed higher NF expression that those of males. On the other hand, NF expression was not significantly associated with tumor recurrence, age, lymph node involvement, tumor grade and tumor stage or disease outcome. Conclusion: Positive NF expression in CRC is a sign of aggressive disease and poor outcome.

Colorectal cancer (CRC) accounts for high mortality. So far, there is lack of markers capable of predicting which patients are at risk of aggressive course of the disease. Protein phosphatase‐2A (PP2A) inhibitor proteins have recently gained interest as markers of more aggressive disease in certain cancers. Here, we report the role of PP2A inhibitor PME‐1 in CRC. PME‐1 expression was assessed from a rectal cancer patient cohort by immunohistochemistry, and correlations were performed for various clinicopathological variables and patient survival. Rectal cancer patients with higher cytoplasmic PME‐1 protein expression (above median) had less recurrences (P = 0.003, n = 195) and better disease‐free survival (DFS) than the patients with low cytoplasmic PME‐1 protein expression (below median). Analysis of PPME‐1 mRNA expression from TCGA dataset of colon and rectal adenocarcinoma (COADREAD) patient cohort confirmed high PPME1 expression as an independent protective factor predicting favorable overall survival (OS) (P = 0.005, n = 396) compared to patients with low PPME1 expression. CRC cell lines were used to study the effect of PME‐1 knockdown by siRNA on cell survival. Contrary to other cancer types, PME‐1 inhibition in CRC cell lines did not reduce the viability of cells or the expression of active phosphorylated AKT and ERK proteins. In conclusion, PME‐1 expression predicts for a favorable outcome of CRC patients. The unexpected role of PME‐1 in CRC in contrast with the oncogenic role of PP2A inhibitor proteins in other malignancies warrants further studies of cancer‐specific function for each of these proteins.

To be effective in vivo, antisense oligonucleotides (AS ON) should be nuclease resistant, form stable ON/RNA duplexes and support ribonuclease H mediated heteroduplex cleavage, all with negligible non-specific effects on cell function. We report herein that AS ONs containing a 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) sugar modification not only meet these criteria, but have the added advantage of maintaining high intracellular concentrations for prolonged periods of time which appears to promote longer term gene silencing. To demonstrate this, we targeted the c-MYB protooncogene's mRNA in human leukemia cells with fully phosphorothioated 2'F-ANA-DNA chimeras (PS-2'FANA-DNA) and compared their gene silencing efficiency with AS ON containing unmodified nucleosides (PS-DNA). When delivered by nucleofection, chemically modified ON of both types effected a90% knockdown of c-MYB mRNA and protein expression, but the PS-2'F-ANA-DNA were able to accomplish this at 20% of the dose of the PS-DNA, and in contrast to the PS-AS DNA, their silencing effect was still present after 4 days after a single administration. Therefore, our data demonstrate that PS-2'F-ANA-DNA chimeras are efficient gene silencing molecules, and suggest that they could have significant therapeutic potential.

BACKGROUND For this study, the diagnostic protocol included fine-needle aspiration biopsy (FNAB) and DNA cytometric measurements. The objective of the study was to improve the diagnostic sensitivity of FNAB. METHODS Sixty-eight FNAB samples were stained using the Feulgen stain, and DNA histograms were produced by selecting the nuclei of cell groups and free cells separately. RESULTS Among 28 samples that were classified as definitely benign (n = 17 samples) or atypical but benign (n = 11 samples), DNA cytometry generally showed diploid/peridiploid peaks. Three of those samples were associated later with carcinoma. In those samples DNA cytometry did not improve sensitivity. Among moderately atypical samples (n = 17 samples), 8 samples were diagnosed later as carcinoma. DNA cytometry helped the diagnostic procedure in 62.5% of those samples. Among 21 samples that were classified as highly suspicious (n = 10 samples) or definitely malignant (n = 11 samples), DNA cytometry generally showed atypical DNA histograms (20 of 21 histograms), and DNA cytometry supported the diagnosis of carcinoma in 95.2%. Histograms that were based on free cells frequently were more abnormal compared with histograms that were based on cell groups. Histologically verified benign lesions also could show abnormalities in DNA histograms. Accepting wider gates than are used normally for primarily Feulgen stained samples on these restained samples resulted in improved specificity, efficiency, and predictive values. CONCLUSIONS DNA cytometry has a potential to support the differential diagnosis of breast lesions, and sampling of free cells increases sensitivity. Benign breast lesions (fibrocystic disease, fibroadenoma) included DNA-cytometrically abnormal cell clones and showed tendencies toward polyploidy, which should be included in the diagnostic criteria. Cancer (Cancer Cytopathol) 2004. © 2004 American Cancer Society.

48 fine needle aspiration biopsy (FNAB) samples from 25 breast cancer cases, originally used for cytodiagnosis were subjected to DNA cytometry. There were air dried smears stained with the MGG method, and samples stained with HE or PAP stain after 50% ethanol fixation and cytocentrifugation. Different sampling strategies were applied. Four methods were tested: method 1: cell groups measured, method 2: all cells measured, method 3: free cells measured, and method 4: atypical free cells measured. Method 4 showed most often DNA aneuploid histogram patterns, sampling method 1 had the highest number of DNA diploid histogram patterns. Diagnostic approaches may benefit from a sampling method detecting the hiding aneuploid cell population. Grading of neoplasm could potentially benefit from other approaches.

Despite compelling evidence from the genetic background and clinical studies indicating that cyclooxygenase-2 (COX2) up-regulation is a key step in carcinogenesis of colorectal carcinoma (CRC), controversy regarding its role as a prognostic factor exists. However, all evidence indicates that increased COX2 activity promotes progression of CRC. This study, aimed to evaluate the expression of COX2 in CRC, and correlate it with different patient clinicopathological data, emphasizing on the role of COX2 as a prognostic factor for CRC.In the present study, archival samples from 145 patients with stage I, II, III, or IV CRC treated during 1981-1990 at the Turku University Hospital (Finland) were used (as microarray blocks) to analyze COX2 expression by immunohistochemistry (IHC).Higher levels of COX2 expression were associated with higher TNM class (p0.06), and higher Dukes' stage (p0.045). In contrast, there was no significant correlation with age, gender, tumor grade or lymph node status. However, univariate survival analysis of metastases showed borderline association with COX2 expression in that patients with metastases with COX2-positive tumors were alive for shorter periods of time compared with patients whose tumors had no COX2 expression (p0.023, log-rank).COX-2 expression has shown a significant correlation with tumor stage and hence is assumed to be a prognostic factor in our cohort of colorectal cancer patients.

Clinical staging and histological grading after surgery have been the “gold standard” for predicting prognosis and planning for adjuvant therapy of colorectal cancer (CRC). With the recent development of molecular markers, it has become possible to characterize tumors at the molecular level. This is important for stage II and III CRCs, in which clinicopathological features do not accurately predict heterogeneity, e.g., in their tumor response to adjuvant therapy. In the present study, archival samples from 141 patients with stage I, II, III, or IV CRC treated during 1981–1990 at Turku University Hospital (Finland) were used (as microarray blocks) to analyze MUC2 expression by immunohistochemistry. Altogether, 49.7 % of all tumors were positive for MUC2. There was no significant correlation between MUC2 expression and age (P

The traditional staging system is currently inadequate for identifying those patients with colorectal carcinoma (CRC) who carry a high risk for poor outcome. In this study, the expression of E-cadherin was evaluated in CRC to determine its correlation with clinico-pathological variables, and association with disease outcome in patients with long-term follow-up. The present series consisted of tissue samples obtained from 230 patients with stage I, II, III, or IV CRC treated during 1981-1990 at Turku University Hospital. Archival paraffin-embedded samples were used to build up tissue microarray blocks, and E-cadherin expression was assessed by immunohistochemistry using an automated staining system. Different grading systems were tested for expression of E-cadherin. Fifty-nine percent of all tumors were positive for E-Cadherin. There was no significant correlation between E-cadherin expression and gender (p0.83), localization (p0.45), tumor invasion (p0.32), or histologic grade (p0.41). However, loss of E-cadherin expression was significantly associated with older age (p0.03) and lymph node involvement (p0.02), and with borderline significance with advanced stage (p0.09) and tumor metastasis (p0.09). In univariate (Kaplan-Meier) survival analysis, positive E-cadherin significantly (p = 0.009) predicted longer disease-free survival (DFS), and the same was true with disease-specific survival (DSS) as well (p = 0.007). In multivariate (Cox) survival analysis, E-cadherin retained its significance as independent predictor of DFS (HR = 1.56; 95% CI 1.01-2.42, p = 0.043), but not DSS. A sub-group analysis revealed that E-cadherin expression also predicts DFS (p0.01) and DSS (p0.04) in stage II CRC. Our results implicate the usefulness of E-cadherin expression in predicting disease recurrence and long-term survival in CRC.

The sensitivity for identification of malignant cells in conventional fine-needle aspiration biopsy (FNAB) investigation is about 80%. This percentage is dependent on the number of examined cells, type of breast cancer, and experience of the examiner. The aim of our study was to estimate the supporting value of image DNA cytometry of FNAB of the breast, and do so by using different sampling methods.This retrospective study was based on 41 cases with an available histological diagnosis: 18 benign lesions and 23 malignant tumours were examined. The smears were submitted to image DNA analysis in a three-step protocol: (i) smears stained with HE method were destained and (ii) then restained with Feulgen staining for DNA and (iii) finally analysed using image cytometry.All non-malignant cases had diploid histogram. However, a few of them had one or two cells of5c category. Most histologically malignant cases were aneuploid. Only three invasive ductal carcinomas showed diploid histograms. All samples with aneuploid histograms were malignant.The results confirm earlier published data in the Finnish population and indicate that image DNA cytometric analysis of nuclear content is a useful marker for identification of malignant cells in FNAB, especially after free cell sampling. The method can be used to increase the cytological sensitivity and specificity in doubtful breast lesions.

Defects of some DNA polymerases have shown associations with cancer, but data on DNA polymerase epsilon are limited. This study investigated mutations in the 55 kDa subunit gene of DNA polymerase epsilon in colorectal cancer.DNA from 16 human colorectal cancer and 9 control samples was studied with polymerase chain reaction-single-strand comformation polymorphism analysis and DNA sequencing.DNA polymerase epsilon gene alterations were identified in 5 out of the 16 cases (31.2%). Two samples showed a T-C transition at exon 17 (potential tyrosine to histidine substitution), and an A-G transition at intron 7; one sample showed an A-G transition at intron 8. An AATT deletion was observed at intron 18 in 3 out of the 16 colon cancer cases (grades 2, 3, and 2, and Dukes' classes C, D, and C, respectively).Because the AATT deletion has also been found in breast cancer, the region may be a mutation hot spot, possibly involved in the carcinogenetic path in advanced colorectal cancer.

Approximately 30% of all colorectal cancer (CRC) patients are diagnosed with stage II disease. Adjuvant therapy is not widely recommended. However, it is well established that a subgroup of patients with stage II are at high risk for recurrence within their lifetime and should be considered for adjuvant chemotherapy. The present work was designed to study the prognostic value of nuclear DNA content in stage II CRC of patients with long-term followup.Isolated nuclei from 50 microm-thick paraffin sections of tissue samples from 253 patients with stage II CRC, who had undergone bowel resection at Turku University Central Hospital were cytocentrifuged on slides, stained with Feulgen staining, and DNA was measured using a computer-assisted image analysis cytometry system. Different approaches were applied in analysis of DNA histograms.DNA content did not show any relation with age (p0.96), sex (p0.35), tumor invasion (p0.77), or grade (p0.31). Aneuploid DNA content was significantly more frequent in the cancer of the left colon and rectum than the right colon (p = 0.02). S-phase fraction analysis revealed that a higher proportion (62%) of the older patients (65 years) had high proliferation rates than did the younger patients (p0.05). Patients with narrow range histograms had a better disease-free survival (DFS) (narrow range: 70%, wide range: 60% at 10 years). Tumors with9c nuclei were associated with significantly better DFS and disease-specific survival (DSS) as compared with the patients who did not have9c nuclei in their tumor samples (p0.003 and p0.0001, respectively). Multivariate survival (Cox) model showed that only classification of the basic pattern of the histogram [odds ratio OR) = 29.14; 95% confidence interval (CI) 2.350-361.57] (p = 0.009) and recurrence (OR = 165.35; 95% CI 48.42-564.7) (p = 0.0001) proved to be independent predictors of clinical outcome.Our results seem to suggest it truly is possible, by using DNA cytometry, to find groups with different prognosis among stage II cases. Those with a high recurrence rate should be considered for adjuvant chemotherapy.

Nuclear morphometry can be expected to improve the distinction between benign and malignant lesions.Forty fine-needle aspiration biopsy (FNAB) samples fixed in 50% ethanol were collected at the African Oncology Institute, Sabratha, Libya, during the period 2004-2007. All diagnoses reported were confirmed histologically. There were 23 cases of infiltrating ductal carcinoma and 17 of benign breast disease. Two different assessment methods were applied: measurements made on cell groups, and those made on free cells. Apocrine metaplasia was excluded. Five different size parameters (include mean nuclear area, MNA) and 6 shape factors were measured.The size parameters showed significant differences between benign and malignant cases. The mean, median and 95% percentiles of nuclear area in both types of assessment were especially significant. The shape parameters were not significant.The study suggests that interactive computerized nuclear morphometry is an efficient and successful tool in distinguishing between cases of benign and malignant disease. Combination of our data with earlier free cell data gave the following diagnostic guidelines: Range of overlap in free cell samples: MNA 55 microm2 - 71 micro2. Cut-off values for diagnostic purposes: 100% detection of malignant cases: MNA54 microm2 (specificity 84%), 100% detection of benign cases: MNA72 microm2 (sensitivity 91%).

AIM: To investigate the changing pattern of α-catenin expression and its relationship to clinical and pathological features of colorectal cancer (CRC) patients. METHODS: Archival tumor samples were analyzed using immunohistochemistry (IHC) for α-catenin in 91 patients with advanced CRC. RESULTS: The values of α-catenin membrane index (MI) and cytoplasmic index (CI) were significantly related to the depth of tumor invasion (P = 0.027, P = 0.020, respectively), high indices being associated with increased depth of the primary tumor invasion (T3 and T4). Similarly, patients with high α-catenin expression had a significantly increased risk of lymph node metastasis (32/39 vs 37/52 for MI and 37/45 vs 32/46 for CI) (P = 0.001, P = 0.0001, respectively, for LNN status). An altered expression (i.e., cytoplasmic pattern) was also related (P = 0.047) to the response to chemotherapy; patients with low CI were more responsive (CR: 7/46) than patients with high CI values (CR: 0/45). There was a marginal effect on survival in patients time with metastases (SWM) (P = 0.087); patients with low CI showing slightly longer SWM, but no such effect on disease free survival (DFS) or disease specific survival (DSS). As to co-expression with another member of the adhesion complex (β-catenin), high α-catenin/β-catenin MI index was of marginal significance in predicting longer DSS (P = 0.063, log-rank). CONCLUSION: The results implicate that high α-catenin expression is intimately involved in the key regulatory mechanisms leading to invasive phenotype, lymph node metastases, and progressive disease in CRC.

To study the dynamic events leading to impaired cell-cell adhesion upon transition to the invasive phenotype of colorectal cancer (CRC), we examined three distinct beta-catenin expression patterns (membranous, cytoplasmic, and nuclear) in the paired samples of the primary tumours (P) and their metastatic lesions (M). beta-catenin expression was detected by immunohistochemistry (IHC) in 33 pairs of the primary CRC and their metastases. In a pair-wise (P-M) comparison, the membranous index (MI) was significantly different between P and M (p=0.036, Wilcoxon Signed-Ranks test), while cytoplasmic index (CI) and nuclear index (NI) values did not significantly deviate between P and M. MI in primary tumours was inversely related to the patient's age (p=0.04) and tumour grade (p=0.03), while patients with low MI in M had a high rate of metastasis at diagnosis (p=0.06). CI in P was lower in patients with LN involvement (p=0.02) and in advanced tumour stage (p=0.002). Tumours of the ascending colon had the highest CI in their M (p=0.04). Interestingly, high MI of the M lesions was a significant predictor of favourable overall survival (OS) in univariate (Kaplan-Meier) survival analysis (p=0.035). In conclusion, significant aberrations in beta-catenin expression probably take place in CRC cells during the development of metastatic phenotype, but a change from membrane expression to cytoplamic and/or nuclear expression is not a prerequisite for metastasis in all cases.

Ezrin is a membrane-cytoskeleton anchor, which, in experimental models, regulates tumor cell invasion and metastatic ability. We carried out immunohistochemical analysis of ezrin in 74 advanced colorectal cancer patients and correlated it to clinicopathologic variables and disease outcome. In contrast to the predominantly membraneous immunoreactivity of normal colorectal epithelium, ezrin expression in the colorectal cells was typically cytoplasmic. Altogether, 16.2% (12/74) of the tumors showed negative/weak ezrin staining, 35.1% (26/74) had moderate staining, and 48.6% (36/74) had intense staining. The expression was more intense in colon than in rectal carcinomas (P = .003). Increased ezrin expression was associated with adverse outcome, that is, shorter disease-specific survival; 48.3 months and 36.6 months for negative-weak versus intense expression (P = .041) as well as shorter survival with metastases at 36 months (P = .030); the metastases(36) rates in ezrin(neg/weak), ezrin(moderate), ezrin(intense) are 58.3%, 25.0%, and 18.4%, respectively. In univariate survival analysis, dichotomized (negative/weak versus moderate/strong) ezrin expression significantly predicted both the 5-year disease specific survival (P = .035) and 5-year metastases (P = .018) but lost this predictive power in multivariate (Cox) analysis. High ezrin expression was also related to high E-cadherin (cytoplasmic) expression, DNA aneuploidy, and high thymidylate synthase expression (P = .046, P = .042, P = .046, respectively). These results suggest that ezrin may play a role in colorectal cancer progression and that ezrin expression might provide clinically valuable information in predicting the biological behavior of colorectal cancer.

Thymidylate synthase (TS) is the rate-limiting enzyme in the synthesis of pyrimidine nucleotides and as such a critical target for fluoropyrimidines, which are widely used in the treatment of colorectal cancer (CRC). The purpose of this study was to investigate TS expression in the primary tumours (PTs) and their metastases (M) in advanced CRC.TS expression was determined immunohistochemically in paraffin-embedded biopsies of PT-M pairs in 39 CRC patients, as related to the clinical data.There was no difference in the mean TS index of PTs compared with that of M, 1.25 and 1.14, respectively (p=0.12). TS expression of PTs was above the mean more often than that of M (61.5% and 41.0%, respectively, p=0.035). High TS expression in PTs was significantly related to high expression in M (the Fisher exact test, p=0.001). Using the absolute index values, TS expression in PT and M was significantly correlated (Pearson R=0.501, p=0.001). In 29/39 (74.3%) pairs, PT and M had concordant expression levels (Cohen's kappa 0.508, 95% CI 0.260-0.756, p=0.001; intraclass correlation coefficient (ICC) = 0.679, 95% CI 0.358-0.836, p=0.0001). No significant association was found between TS expression and any of the clinicopathological variables, disease outcome (DFS, DSS) or its response to treatment in univariate or multivariate analysis.Albeit usually higher, TS expression in PT was closely correlated with TS expression in M. This suggests that measurement of TS in primary CRC accurately predicts TS expression in subsequent metastases, which may help in selecting those patients most likely to respond to 5-FU-based regimens.

A better understanding on the development of a metastatic phenotype in colorectal cancer (CRC) is essential to help identify patients at high risk for metastasis. Therefore, we have studied the role of the CD44 family of trans-membrane glycoprotein in the process of CRC metastasis, by examining the expression of CD44s and CD44v6 in primary tumours and their metastatic lesions in 46 patients using immunohistochemistry. The expression of both CD44s and CD44v6 was significantly higher (moderate/strong) in primary tumours as compared to their metastases (p=0.008, p=0.0001, respectively). CD44s expression in metastases increased with the degree of the histological grade (p=0.009) and invasiveness of the primary tumour (p=0.002). Disease-free survival (DFS) was shorter in patients who had metastases with a strong/moderate expression of CD44s as compared to those with negative/weak expression (8.3 months vs 16.9 months p=0.221, respectively). Our finding that CD44s expression in metastatic lesions may reflect the aggressiveness of the primary tumour from which it has originated implicates an important link between the two lesions. CD44 expression may also provide valuable biological information as suggested by the observation that up-regulated CD44s expression in metastases is associated with a shorter DFS.

Thymidylate synthase (TS) is the rate-limiting enzyme in the synthesis of pyrimidine nucleotides, required for DNA synthesis, and is also a critical target for fluoropyrimidines, which are widely used in the treatment of gastrointestinal tumours. We examined TS expression in tumours from 86 patients with advanced colorectal cancer who received one of two chemotherapy regimes (either irinotecan alone or irinotecan and 5-flurouracil with folinic acid). TS expression was determined immunohistochemically in 86 paraffin-embedded primary tumour sections and assessed using image analysis software. TS was significantly associated with survival, with lower levels of TS expression associated with longer patient survival (p=0.02). Patients with an objective response to treatment had a longer median survival than those who did not show an objective response to treatment (p=0.001). A significant association was found between tumour TS expression and response to treatment with 5-FU plus FA with irinotecan (p=0.05). Sixty-four percent of patients with TS expression levels below the median showed an objective (complete or partial) response to treatment while, 38% with TS expression levels above the median responded. Immunohistochemical TS expression levels might be used both as a prognostic marker and to help identify patients who would benefit from 5-FU based regime.

Cell adhesion molecules (CAMs) are cell surface glycoproteins that are important in cell-cell and cell-matrix interactions and play an important role in cell growth and differentiation. We examined immunohistochemically CD44s, CD44v6 and E-cadherin expression in 86 formalin-fixed, paraffin-embedded primary tumours and 5 metastases. Lower levels of CD44s, CD44v6 and membranous E-cadherin expression were significantly associated with higher tumour grade (p=0.022, p=0.016 and p= 0.041, respectively). Moreover, CD44v6 and membranous E-cadherin expression were correlated with the depth of primary tumour invasion (p=0.030 and p=0.020, respectively), and increased expression of CD44v6 and decreased membranous E-cadherin expression were associated with increased primary tumour invasion. The results suggest that these CAMs are associated with tumour differentiation and invasion in locally advanced and metastatic colorectal carcinoma.

The aim of this study was to investigate the potential of computerized nuclear morphometry and DNA image cytometry in characterizing the apocrine change of mammary epithelium in fine-needle aspiration biopsy (FNAB). The effect of two different sample processing techniques on the results was also studied. Mean nuclear areas in air-dried smears ranged from 59.0 microm2 to 151.0 microm2 and in ethanol-fixed samples from 32.3 microm2 to 63.4 microm2. The DNA histograms of apocrine cells usually showed a dominant peak in the diploid region. In some cases the mode of the peak was slightly shifted to the right or left in respect to the control peak. One case had a tetraploid cell population, suggesting atypical apocrine change. After histological investigation this case was diagnosed as infiltrating carcinoma. The patient had earlier been treated with x-ray irradiation for a mediastinal lymphoma. Findings of nuclear morphometry and DNA cytometry in apocrine metaplasia are here described in a systematic study for the first time. The data suggest that these methods may help in distinguishing premalignant and malignant apocrine lesions from typical apocrine metaplasia of mammary epithelial cells.

To study the potential of nuclear morphometry in supporting the interpretation of fine needle aspiration biopsy (FNAB) samples of the breast fixed in 50% ethanol and centrifuged on slides.Computerized morphometry was used to outline the nuclei of breast epithelial cells in breast cancer, fibroadenoma and fibrocystic disease. The diagnoses were histologically confirmed. We applied 2 different sampling methods (measurements done on cell groups and on free cells).The mean nuclear area of cell groups of malignant samples (23) varied from 42 to 125 microns 2, in fibroadenomas from 30 to 50 microns 2 and in fibrocystic disease from 26 to 57 microns 2. The mean nuclear area of free cells varied as follows: cancer, 66-181 microns 2; fibroadenoma, 33-70 microns 2; fibrocystic disease, 35-60 microns 2. Apocrine metaplasia was excluded from comparison on a morphologic basis.The study suggests that if the mean nuclear area of cell groups is42 microns 2, the lesion is probably benign; if57 microns 2, and apocrine metaplasia is excluded, malignancy should be considered. The differential diagnosis between carcinoma and fibroadenoma could be based on free cells: mean area of free cell nucleior = 65 microns 2 suggested a benign lesion, and ofor = 71 microns 2 suggested a malignant lesion. Morphometric nuclear size features (exemplified by nuclear area) appeared efficient in distinguishing between malignant and benign lesions when measured from free cells and cell groups.

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.

We tried to improve the evaluation of E-cadherin immunostaining in paraffin sections, to distinguish the less aggressive variants of ductal infiltrating breast cancer from other variants.The method graded the membrane staining and estimated the fraction of area of cancer tissue stained at the respective staining grade, resulting in an immunohistochemical staining index. At the cut-point 0.35 the index divided all 157 patients (P=0.0188), and 57 node-positive patients (P= 0.0006) into two groups of different survival. In multivariate analysis (all patients) E-cadherin immunoscore was inferior to mitotic index (SMI) (P=0.0002), but still significant (P=0.0031). Among node-positive patients E-cadherin was even more powerful and superior (P=0.0001) to the still significant SMI (P=0.0023), and E-cadherin immunostaining and the mitotic activity (SMI) combined did not need the support of other prognosticators in the Cox model.The study suggests that E-cadherin immunostaining can be used efficiently in finding patients with favourable outcome among node-positive patients.

AIM: To investigate the changing pattern of β-catenin expression and its prognostic value in advanced colorectal cancer (CRC). METHODS: Archival tumor samples were analyzed for β-catenin using immunohistochemistry (IHC) in 95 patients with advanced CRC. RESULTS: Membranous β-catenin expression was found in the normal colorectal epithelium. Almost 100% of CRC cases showed membranous and cytoplasmic expression, and 55 (58%) cases showed nuclear expression. In univariate (Kaplan-Meier) survival analysis, only the nuclear index (NI) was a significant predictor of disease-free survival (DFS) (P = 0.023; n = 35), with a NI above the median associated with longer DFS (34.2 mo) than those with a NI below the median (15.5 mo) (P = 0.045, ANOVA). The other indices were not significant predictors of DFS, and none of the three tested indices (for membranous, cytoplasmic, or nuclear expression) predicted disease-specific survival (DSS). However, when dichotomized as positive or negative nuclear expression, the former was a significant predictor of more favorable DFS (P = 0.041) and DSS (P = 0.046). CONCLUSION: Nuclear β-catenin expression provides additional information in predicting patient outcome in advanced CRC.

AIM: To investigate the changes that occur in E-cadherin expression during the process of metastasis in colorectal cancer. METHODS: E-cadherin expression was detected by immunohistochemistry and two indices of expression were calculated which reflected the level of expression and the locations (membrane and cytoplasm). Univariate and multivariate survival analyses were used to assess the value of these two E-cadherin indices as predictors of both disease-free (DFS) and disease-specific (DSS) survival. RESULTS: E-cadherin membrane index (MI), but not cytoplasmic index (CI), was significantly higher in primary tumors than their metastases (P = 0.0001). Furthermore, both primary tumor MI and CI were higher among the patients who developed subsequent metastasis (P = 0.022 and P = 0.007, respectively). Interestingly, both indices were higher in liver metastase compared to other anatomic sites (MI, P = 0.034 and CI, P = 0.022). The CI of the primary tumors was a significant predictor of DFS (P = 0.042, univariate analysis), with a strong inverse correlation between CI and DFS (P = 0.006, multivariate analysis). Finally, the MI of primary tumor proved to be a significant independent predictor of DSS, with higher indices being associated with a more favorable outcome (P = 0.016). CONCLUSION: Examination of E-cadherin expression and distribution in colorectal tumors can be extremely valuable in predicting disease recurrence. The observation that aberrant cytoplasmic expression of E-cadherin can predict disease recurrence is obviously of great importance for both patients and clinicians, and significantly affects decisions concerning the therapy and management of the patients.