Children with congenital colorectal malformations during the UK Sars-CoV-2 pandemic lockdown: an assessment of telemedicine and impact on health.

PURPOSE: This pilot study was designed to assess bowel function and quality of life (QoL) in children and adolescents with congenital colorectal malformations (CCM) during the first UK COVID lockdown period. METHODS: Changes in health were assessed through semi-structured interviews, gastrointestinal functional outcomes using Krickenbeck scoring and QoL by the modified disease-specific HAQL (Hirschsprung's disease anorectal malformation quality of life questionnaire). The State-Trait Anxiety Inventory (STAI)™ for adults was used to assess parental anxiety. RESULTS: Thirty-two families were interviewed; 19 (59%) reported no change in their child's health during the lockdown, 5 (16%) a deterioration and 8 (25%) an improvement. Neither the severity of the CCM, nor the degree of bowel dysfunction, correlated with any deterioration. The HAQL score was not correlated to a change in health. Anxiety scores ranged from no anxiety to clinical concerns. Telemedicine was well accepted by 28/32 parents (88%); however, in-person appointments were preferred if there were clinical concerns. CONCLUSION: In the follow-up of children and adolescents with CCM during the first UK lockdown using telemedicine we found that over half had stable health conditions. Patients needing additional care could not be predicted by the severity of their disease or their bowel function alone.

Association of BRCA1, ERCC1, RAP80, PKM2, RRM1, RRM2, TS, TSP1, and TXR1 mRNA expression levels between primary tumors and infiltrated regional lymph nodes in patients with resectable non-small cell lung cancer.

Differences in gene expression levels between the primary tumors (PTs) and matched regional lymph nodal metastases (LNs) in patients with totally excised non-small cell lung cancer (NSCLC) were explored. Microdissected formalin-fixed paraffin-embedded (FFPE) samples from (PT) and their matched infiltrated LNs, from 239 patients [183 (with matched PT and LNs samples)-case and 56 PT only samples-control cohorts] were analyzed for BRCA1, ERCC1, RAP80, PKM2, RRM1, RRM2, TS, TSP1, and TXR1 mRNA expression by quantitative real-time polymerase-chain reaction (PCR). Moderately positive correlation between the expression of each gene in the PT and the matched LNs was observed. Concordance rates between the PT and the LNs were: BRCA1 (67.7%), ERCC1 (68.4%), PKM2 (63.4%), RAP80 (68.8%), RRM1 (70.9%), RRM2 (69%), TS (72.9%), TSP1 (69.8%), TXR1 (63.7%). Expression levels and their differences were correlated with Relapse-Free Survival (RFS) and Overall Survival (OS). High BRCA1 PT in patients with squamous histology was associated with increased OS (p = 0.036). High TSP1 PT levels were shown to be the only independent prognostic factor for OS and RFS (p = 0.023 and p = 0.007). PKM2 low levels in both PT and matched LNs were associated with better OS irrespective of the underlying histology (p = 0.031). RRM1 discordant levels between PT and matched LNs were associated with worse OS in squamous tumors (p = 0.019) compared to patients with both low expression in PT and LN.TXR1 high levels in both PT and matched LNs were associated with better OS in patients with squamous tumors (p = 0.007).These findings indicate that there is different gene expression between PT and matched LNs which may affect the outcome in early NSCLC and therefore PT's molecular biology should not be the sole determinant for prognostication.

The presence of Merkel cell polyomavirus is associated with deregulated expression of BRAF and Bcl-2 genes in non-small cell lung cancer.

Polyomaviruses such as BK virus (BKV), JC virus (JCV) and Merkel cell polyomavirus (MCPyV) are typically nononcogenic, although they have been detected in a variety of human neoplasms. The aim of our study was to determine the frequency of the most common polyomaviruses MCPyV, BKV and JCV as well as the gene expression profile of genes involved in oncogenesis including K-ras, BRAF, RKIP, Bax, Bcl-2, p53 and RB1 in a cohort of non-small cell lung cancer (NSCLC) patients. Real-time and nested polymerase chain reaction (PCR) were used to assess the presence of polyomaviruses DNA in tissue biopsies from 110 patients with primary NSCLC and 14 tissue specimens from macroscopically healthy sites of their lung. Real-time PCR was also used to determine the mRNA expression of K-ras, BRAF, RKIP, Bax, Bcl-2, p53 and RB1 in selected samples. Results showed that ten NSCLC specimens were positive for the presence of MCPyV DNA (10/110, 9.1%), whereas no control sample was tested positive for the virus. The MCPyV-positive samples were predominantly obtained from male smokers (9/10). BKV and JCV DNA were not detected either in lung tissues biopsies or the control specimens. Interestingly, gene expression analysis revealed increased mRNA and protein expression of BRAF gene in association with BRAF phosphorylation in the MCPyV-positive samples, whereas Bcl-2 gene expression was downregulated in the same type of samples. The detected MCPyV prevalence in NSCLC in combination with the deregulated expression of BRAF and Bcl-2 genes suggests that these events are likely to contribute to the pathogenesis of NSCLC.

Preoperative assessment of neurovesical function in children with anorectal malformation: association with vertebral and spinal malformations.

PURPOSE: We preoperatively assessed neurovesical function and spinal cord function in children with anorectal malformations. In cases of neurovesical dysfunction we looked for an association with vertebral malformation or myelodysplasia. MATERIALS AND METHODS: We prospectively evaluated 80 children with anorectal malformations via preoperative urodynamics and magnetic resonance imaging of the spine. Bladder compliance and volume, detrusor activity and vesicosphincteric synergy during voiding allowed urodynamic evaluation. Results were reported according to Wingspread and Krickenbeck classifications of anorectal malformations. RESULTS: Urodynamic findings were pathological in 14 children (18%). Pathological evaluations did not seem related to type of fistula or level of anorectal malformation. Vertebral anomalies were seen in 34 patients (43%) and myelodysplasia in 16 (20%). Neither vertebral anomaly nor myelodysplasia seemed associated with type of fistula or severity of anorectal malformation. Of 14 children with pathological urodynamics no vertebral anomaly or myelodysplasia was found in 7. Of 66 children with normal urodynamics 40 presented with vertebral or spinal malformation. CONCLUSIONS: Lower urinary tract dysfunction is common in patients with anorectal malformations. Normal spine or spinal cord does not exclude neurovesical dysfunction. Myelodysplasia or vertebral anomaly does not determine lower urinary tract dysfunction. Thus, we recommend preoperative urodynamic assessment of the bladder and magnetic resonance imaging of the spine in children with anorectal malformations.

Infliximab restores the balance between pro- and anti-apoptotic proteins in regressing psoriatic lesions.

BACKGROUND: Psoriasis and psoriatic arthritis are treated very efficaciously with infliximab, a chimaeric human-murine antitumour necrosis factor (TNF)-α antibody. As we reported earlier, infliximab, besides its anti-inflammatory properties, induces a caspase-independent programmed cell death of psoriatic keratinocytes. OBJECTIVES: To elucidate this finding further, we investigated the epidermal expression of proteins involved in the mitochondria-dependent (intrinsic) pathway of cell death. METHODS: Quantification of proteins with pro- (p53, AIF, Bax) and anti-apoptotic functions (Bcl-2, Bcl-XL) and of NF-κB was performed by means of immunohistochemistry and digital image analysis of the staining of nonlesional skin and lesional psoriatic skin from patients treated with infliximab at weeks 0, 2 and 6. RESULTS: Serial biopsies from psoriatic plaques of samples taken at days 0, 5, 14 and 21 of therapy demonstrated a significant downregulation of anti-apoptotic proteins Bcl-2, Bcl-XL and NF-κB during treatment and, in parallel, a significant upregulation of pro-apoptotic proteins p53, Bax and AIF. These differences in expression correlated with decreases in epidermal thickness and clinical outcome (Psoriasis Area and Severity Index). At day 21, expression levels of apoptosis-related proteins in lesional skin approximated those found in nonlesional skin. CONCLUSIONS: Our data therefore suggest that TNF-targeting agents may induce the regression of psoriasis at least in part by normalizing the expression of apoptosis-related proteins in lesional keratinocytes.

Cytopathologic interpretation of ascites due to malignancy.

The diagnosis of metastatic cancer in peritoneal fluid is of great importance for the patient and the attending physician. A cytopathologist's responsibility is twofold: (1) to accurately identify malignant cells; (2) to interpret tumor type and if possible the site of its origin even in the absence of complete clinical history of other clues. The difficulty in the diagnosis of metastatic neoplasms in peritoneal fluid is due to 2 factors: (1) abnormal mesothelial cells or macrophages may simulate cancer cells, or may conceal tumor cells; and (2) peritoneal fluid constitutes a natural and hitherto inadequately explored medium of cell culture, in which neoplastic cells may proliferate free of the boundaries imposed upon them by the framework of organs and tissues. Immunocytochemistry (ICC) and molecular techniques are essential to establish an accurate diagnosis. From a great many points of view malignant peritoneal fluid is suitable for continuous study of cancer cells, thus providing knowledge about biologic aspects of human solid tumors.

Correlation of BRCA1, TXR1 and TSP1 mRNA expression with treatment outcome to docetaxel-based first-line chemotherapy in patients with advanced/metastatic non-small-cell lung cancer.

BACKGROUND: We explored the predictive significance of BRCA1, TXR1 and TSP1 expression in non-small-cell lung cancer (NSCLC) patients treated with docetaxel in association with cisplatin or gemcitabine. METHODS: To analyse BRCA1, TXR1 and TSP1 mRNA expression from microdissected primary tumours of 131 patients with stage IIIB (wet) and IV NSCLC, RT-qPCR was used. RESULTS: The mRNA levels of TXR1/TSP1 were inversely correlated (Spearman's test: -0.37; P=0.001). Low TXR1 mRNA levels were associated with higher response rate (RR; P=0.018), longer median progression-free survival (PFS; P=0.029) and median overall survival (mOS P=0.003), whereas high TSP1 expression was correlated with higher RR (P=0.035), longer PFS (P<0.001) and mOS (P<0.001). Higher BRCA1 mRNA expression was associated with higher RR (P=0.028) and increased PFS (P=0.021), but not mOS (P=0.4). Multivariate analysis demonstrated that low TXR1/high TSP1 expression was an independent factor for increased PFS (HR 0.49; 95% CI 0.32-0.76; P<0.001) and mOS (HR 0.37; 95% CI 0.2-0.58; P<0.001), whereas high BRCA1 expression was correlated with increased PFS (HR 0.53; 95% CI 0.37-0.78; P=0.001). CONCLUSIONS: These data indicate that TXR1/TSP1 and BRCA1 expression could be used for the prediction of taxanes' resistance in the treatment of NSCLC.

Monitoring serum levels ELR+ CXC chemokines and the relationship between microvessel density and angiogenic growth factors in multiple myeloma.

BACKGROUND: The ELR+ CXC chemokines are important mediators of tumorigenesis, related to their angiogenic properties. Angiogenesis appears to be a prominent feature in the progression of multiple myeloma (MM). CXC chemokines have four highly conserved cysteine amino acid residues, with the first two cysteine molecules separated by a single amino acid. The angiogenic potential of this group is determined by the presence of three amino acid residues (Glu-Leu-Arg: the ELR motif) preceding the first cysteine amino acid, in the NH2 terminus. AIMS: The purpose of this study was to determine serum concentrations of angiogenesis-related chemokines ELR+ motif, such as interleukin-8 (IL-8), epithelial neutrophil activating protein-78 (ENA-78) and growth-related gene alpha (GRO-α), as well the bone marrow microvascular density (MVD) in patients with MM at diagnosis and after treatment, in plateau phase. We also evaluated the relationship among them with other known growth factors involved in angiogenesis. METHODS: Serum levels of the ELR+ CXC chemokines: IL-8, ENA-78 and GRO-α as well as of the angiogenic factors: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α) were determined in 63 newly diagnosed MM patients, in 30 in plateau phase and in 20 healthy controls. Serum measurements of them were performed with commercially available kits for ELISA. Bone marrow biopsies were performed before and after treatment, in plateau phase, in order to determine MVD by staining vessels with anti-CD31. RESULTS: Serum concentrations of IL-8, ENA-78, GRO-α and TNF-α were significantly higher in the group of MM patients (44.5±25.3, 765±572.1, 186.5±129.1 and 4.2±2.8 pg/ml, respectively) in comparison to control group (27.3±6.4, 335.1±268.6, 112.5±76.1 and 1.3±0.8 pg/ml) (p<0.02 for GRO-α, p<0.001 for other cases). We also found that untreated patients had higher levels of IL-8, ENA-78, GRO-α than post treatment patients, but statistical significant difference was found only for IL-8 (48.36±30.93 pg/ml vs. 35.05±19.77 pg/ml, p<0.001). Furthermore IL-8, GRO-α, TNF-α, HGF and VEGF were significantly higher with increasing disease stage (p<0.001 in all cases). ENA-78 serum levels were higher in stage III than in stage I and II, but without statistical significance. Additionally we correlated each proinflammatory cytokine with well known angiogenic factors such as HGF, VEGF and TNF-α. A positive correlation was found between serum HGF and IL-8 and GRO-α (r=0.316 p<0.01, r=0.297 p<0.02, respectively). Similarly serum VEGF correlated with ENA-78 and GRO-α (r=0.323 p<0.01, r=0.469 p<0.001, respectively). In the pretreatment group of patients a positive correlation between bone marrow MVD and serum levels of GRO-α was found (r=0.304 p<0.01). There was a difference in survival times between patients with higher than median versus low IL-8, ENA-78 and GRO-α levels, but the differences could not reach statistical significance in either case. CONCLUSIONS: These findings support the hypothesis that ELR+ motif CXC chemokines, such as IL-8, ENA-78 and GRO-α correlate with angiogenic growth factors and may play a role in the progression of MM. Further studies are needed to determine their prognostic and predictive significance.

Apoptosis and cell proliferation correlated with tumour grade in peritoneal fluids of patients with serous ovarian cancer.

OBJECTIVE: Apoptosis and cell proliferation in peritoneal fluids of patients with ovarian serous adenocarcinoma have not been well described in cytology. To investigate the contribution of cell death to the growth of this tumour we analysed both apoptosis and cell proliferation in peritoneal fluids of patients with ovarian serous adenocarcinoma. METHODS: We studied 40 tumours from 40 patients with ovarian serous adenocarcinoma. Twelve tumours were high grade, 13 were moderately differentiated and 15 were poorly differentiated. The detection of DNA fragments in situ using the terminal deoxyribonucleotidy transferase (TDT)-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay was applied to investigate active cell death (apoptosis), and the MIB-1 antigen was used to investigate cell proliferation. RESULTS: The TUNEL indices were 0.29 ± 0.05, 0.79 ± 0.10 and 2.1 ± 0.90 in Grade I, Grade II and Grade III ovary carcinomas, respectively. The MIB-1 antigen labelling indices were 6.5 ± 0.09, 12.9 ± 3 and 25.8 ± 6.2, respectively, in the same order of tumour differentiation. The differences in both TUNEL and MIB-1 labelling indices were statistically significant between Grade I, Grade II and Grade III carcinomas and there was a positive correlation between the two indices (P < 0.001). CONCLUSIONS: Apoptosis and cell proliferation increased as the grade of tumour increased in ovarian serous adenocarcinoma, suggesting a rapid turnover of the tumour cells in tumours of higher grade, and may play an important role in the growth and the extension of such cancer cells in the peritoneal cavity.

BRAF mutations, microsatellite instability status and cyclin D1 expression predict metastatic colorectal patients' outcome.

BACKGROUND: The significance of BRAF mutations, microsatelite instability (MSI) status and cyclin D1 expression in patients with metastatic colorectal cancer (mCRC) was evaluated. METHODS: Primary tumours from 144 patients treated for mCRC were assessed for BRAF (V600E) mutation, MSI status and cyclin D1. The data were correlated with progression-free survival (PFS) and overall survival (OS). RESULTS: BRAF mutations were detected in 10 (out of 22, 45%) patients with MSI-H tumours compared with 2 (out of 122, 1.6%) in those with microsatellite stable tumours (P<0.001). The presence of BRAF mutations was correlated with cyclin D1 overexpression (7 out of 26 patients, 58% vs 5 out of 118 patients, 14%; P=0.001). Patients with BRAF-mutated primary tumours had a significantly decreased PFS (2.7 vs 9.8 months; P<0.001) and median OS (14 vs 30 months; P<0.001) than patients with wild-type (wt) tumours. Patients with MSI-H and BRAF-mutated tumours experienced significantly lower PFS (3.1 vs 11.4 months; P=0.008) and OS (14.5 vs 35.5 months; P=0.004) than patients with MSI-H and BRAF wt tumours. Similarly, BRAF mutations and cyclin D1 overexpression were correlated with decreased PFS (3.1 vs 8.6 months; P=0.03) and OS (17.8 vs 39.2 months; P=0.01). CONCLUSION: BRAF V600E mutations are associated with MSI-H status and cyclin D1 overexpression and characterize a subgroup of patients with poor prognosis.

Detection of cytokeratin-19 mRNA-positive cells in the peripheral blood and bone marrow of patients with operable breast cancer.

BACKGROUND: To compare detection rates and evaluate the clinical relevance of cytokeratin-19 (CK-19) mRNA-positive cells in the peripheral blood (circulating tumour cells, CTCs) and bone marrow (disseminated tumour cells; DTCs) of patients with early breast cancer. METHODS: Paired samples of peripheral blood and bone marrow were obtained from 165 patients with stage I-II breast cancer before the initiation of adjuvant chemotherapy. In 84 patients, paired blood and bone marrow samples were also available after chemotherapy. The detection of CK-19 mRNA-positive CTCs and DTCs was assessed by real-time PCR. RESULTS: CK-19 mRNA-positive CTCs and DTCs were detected in 55.2 and 57.6% of patients before chemotherapy, respectively. After chemotherapy, CTCs and DTCs were identified in 44 (52.4%) and 43 (51.2%) of the 84 patients, respectively. There was a 93.9% (McNemar; P=0.344) and 72.6% (McNemar; P=0.999) concordance between blood and bone marrow samples before and after chemotherapy, respectively. The detection of CK-19 mRNA-positive CTCs or DTCs before chemotherapy was associated with decreased overall survival (P=0.024 and P=0.015, respectively). In addition, their simultaneous detection was also associated with an increased incidence of disease-related death and decreased overall survival (P=0.016). CONCLUSIONS: The detection of CK-19 mRNA-positive CTCs using reverse transcription-PCR (RT-PCR) both before and after chemotherapy is correlated with the detection of CK-19 mRNA-positive DTCs in patients with early-stage breast cancer. The determination of the CTC status by RT-PCR conveys clinically relevant information that is not inferior to DTC status and, owing to the ease of sampling, warrants further evaluation as a tool for monitoring minimal residual disease.

Kallikrein 10 (KLK10) methylation as a novel prognostic biomarker in early breast cancer.

BACKGROUND: We evaluated the prognostic significance of KLK10 exon 3 methylation in patients with early-stage breast cancer since it has been shown to have a significant impact on biological characteristics of breast tumors. MATERIALS AND METHODS: Using methylation-specific PCR, we evaluated the specificity of KLK10 methylation in 10 breast tumors and matching normal tissues, 10 breast fibroadenomas, 11 normal breast tissues and in a testing group of 35 patients. The prognostic significance of KLK10 methylation was validated in an independent cohort of 93 patients. RESULTS: KLK10 was not methylated in normal breast tissues and fibroadenomas while it was in 5 of 10 breast tumors and in 1 of 10 matching normal tissues. In the testing group of 35 patients, KLK10 methylation was detected in 70.0% of patients who relapsed (P = 0.001) and in 77.8% of patients who died (P = 0.025). In the independent cohort, 53 of 93 (57.0%) patients were found positive for KLK10 methylation. During the follow-up period, 24 of 93 (25.8%) patients relapsed and 19 of 93 (20.4%) died. Disease-free interval (DFI) and overall survival (OS) were significantly associated with KLK10 methylation (P = 0.0025 and P = 0.003). Multivariate analysis revealed that KLK10 methylation was an independent prognostic factor for DFI and OS. CONCLUSION: KLK10 exon 3 methylation provides important prognostic information in early breast cancer patients.

Ribonucleotide reductase subunits M1 and M2 mRNA expression levels and clinical outcome of lung adenocarcinoma patients treated with docetaxel/gemcitabine.

Ribonucleotide reductase subunits M1 (RRM1) and M2 (RRM2) are involved in the metabolism of gemcitabine (2',2'-difluorodeoxycytidine), which is used for the treatment of nonsmall cell lung cancer. The mRNA expression of RRM1 and RRM2 in tumours from lung adenocarcinoma patients treated with docetaxel/gemcitabine was assessed and the results correlated with clinical outcome. RMM1 and RMM2 mRNA levels were determined by quantitative real-time PCR in primary tumours of previously untreated patients with advanced lung adenocarcinoma who were subsequently treated with docetaxel/gemcitabine. Amplification was successful in 42 (79%) of 53 enrolled patients. Low levels of RRM2 mRNA were associated with response to treatment (P< 0.001). Patients with the lowest expression levels of RRM1 had a significantly longer time to progression (P=0.044) and overall survival (P=0.02) than patients with the highest levels. Patients with low levels of both RRM1 and RRM2 had a significantly higher response rate (60 vs 14.2%; P=0.049), time to progression (9.9 vs 2.3 months; P=0.003) and overall survival (15.4 vs 3.6; P=0.031) than patients with high levels of both RRM1 and RRM2. Ribonucleotide reductase subunit M1 and RRM2 mRNA expression in lung adenocarcinoma tumours is associated with clinical outcome to docetaxel/gemcitabine. Prospective studies are warranted to evaluate the role of these markers in tailoring chemotherapy.

Clinical relevance of circulating CK-19 mRNA-positive cells detected during the adjuvant tamoxifen treatment in patients with early breast cancer.

BACKGROUND: The purpose of this study was to evaluate the effect of adjuvant treatment with tamoxifen on the CK-19 mRNA+ cells in patients with early-stage breast cancer. PATIENTS AND METHODS: CK-19 mRNA+ cells were prospectively and longitudinally detected using a specific real-time PCR assay for CK-19 mRNA in 119 patients with estrogen and/or progesterone receptor-positive tumors during the period of tamoxifen administration. RESULTS: Twenty-two (18.5%) patients had detectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy and in 15 (68.2%) of them adjuvant tamoxifen could not eliminate these cells (persistently positive). In 68 (57.1%) patients, no CK-19 mRNA+ cells could be detected throughout the follow-up period (persistently negative). Seven (46.7%) of the 15 persistently positive and six (8.8%) of the 68 persistently negative patients developed disease recurrence (P = 0.00026). Persistency of CK-19 mRNA+ cells was associated with a significantly lower median disease-free interval (P = 0.0001) and overall survival (P = 0.0005). Multivariate analysis revealed that the detection of CK-19 mRNA+ cells during the administration of tamoxifen was associated with an increased risk of relapse [hazard ratio (HR) = 22.318, P = 0.00006] and death (HR = 13.954, P < 0.00001). CONCLUSIONS: The detection of CK-19 mRNA+ cells throughout the period of adjuvant tamoxifen treatment is an independent poor prognostic factor in patients with early breast cancer.

Expression of endothelial PDGF receptors alpha and beta in breast cancer: up-regulation of endothelial PDGF receptor beta.

The PDGF pathway is essential in tumor angiogenesis. Although the expression of the PDGF receptors has been excessively studied on breast cancer cells, few studies exist on PDGFR expression on the tumor endothelial cells. In the present study, it is investigated whether endothelial PDGF receptors' expression is altered in breast cancer. Endothelial PDGFRalpha and beta expression was initially studied under the influence of tumor conditioned medium derived from a breast cancer cell line. Following tissue culture experiments the endothelial expression of both receptors was studied on formalin-fixed paraffin-embedded tissue sections of normal breast and breast cancer specimens. The tissue culture experiment revealed a possible up-regulation of endothelial PDGFRbeta by breast cancer environment. Immunohistochemistry verified the result since 69.7% of the breast cancer sections were positive for PDGFRbeta compared to 43.3% of normal breast sections (p<0.05). No statistical difference was revealed by studying PDGFRalpha expression. In conclusion, our findings support the thesis of possible anti-PDGFRbeta anti-angiogenic therapy, in cases of endothelial PDGFRbeta-expressing breast cancer.

Protein tyrosine kinase and estrogen receptor-dependent pathways regulate the synthesis and distribution of glycosaminoglycans/proteoglycans produced by two human colon cancer cell lines.

The soy isoflavone genistein can affect cell metabolism by specifically inhibiting protein tyrosine kinase (PTK) and/or interacting with the estrogen receptors (ERs). Glycosaminoglycans (GAG)/proteoglycans (PG) may participate in tumor development and progression. The synthesis of GAG by two human colon cancer cell lines, HT-29 and SW-1116, and the effects of genistein on their production and distribution between culture medium and cell membrane were studied. The mitogenic activity of genistein on both cell lines growth was also examined. Metabolic labeling, sensitive high pressure liquid chromatography (HPLC) techniques and fluorometric cell proliferation assays were utilized. The results demonstrate that both estrogen receptor beta-positive (ERbeta+) cancer cell lines produced hyaluronan (HA), both extracellular and membrane-associated galactosaminoglycans (GalAG) and heparan sulfate (HS), with the HT-29 cells producing all GAG fractions at significantly higher rates. The observed dose-dependent inhibitory effect of genistein on the synthesis of both secreted and cell-associated GAG/PG by the SW-1116 cells, as well as on their growth, was suggestive of a PTK mechanism. On the other hand, the synthesis of GAGs/PGs by HT-29 cells in the presence of genistein was dependent on their type and localization which implies the active participation of the ERs, which was further supported by the observed growth stimulation at low concentrations of genistein.

Correlation of heat shock protein (HSP70) expression with cell proliferation (MIB1), estrogen receptors (ER) and clinicopathological variables in invasive ductal breast carcinomas.

The aim of our study was to evaluate the relationship between the expression of HSP70 protein, cell proliferation, the expression of ER receptors and the clinicopathological variables Grade and LNS in breast invasive human tumors along with the role of HSP70 protein in the prognosis of human breast cancer. A strong association between HSP70 expression and ER content, in agreement with previous data, was found which revealed a statistically significant association between HSP70 positivity and ER expression (p<0.008) in 50 cases of invasive primary human breast cancers. We also found a strong correlation between HSP70 expression, Grade and LNS of invasive ductal breast carcinomas. This suggests that the expression of HSP70 plays a significant role in the progression of human breast cancer, and might prove useful in many other malignancies as an important marker for the outcome of the disease.

Myoepithelial cell cocktail (p63+SMA) for the evaluation of sclerosing breast lesions.

Sclerosing breast lesions may sometimes mimic the appearance of infiltrating carcinoma due to the entrapment of ductular structures in a fibrotic core. The immunohistochemical detection of the outer myoepithelial cell layer that is indicative of a non-infiltrating lesion is a valuable clue for the diagnosis of such ambiguous cases. The myoepithelial cell markers smooth muscle actin (SMA) and p63 are most commonly used since their specificity and sensitivity are well established. However, recent studies have indicated that some morphologically distinct myoepithelial cells fail to stain for SMA and that p63 positivity can be rarely expressed by a subset of malignant epithelial cells. Moreover, SMA can also be positive in stromal myofibroblastic cells and normal vessels that can be found close to the entrapped ductules and might be erroneously interpreted as myoepithelial cells. In this study, we used a double-immunolabeling technique combining both SMA and p63 antibodies (myoepithelial cell cocktail), in order to investigate whether this technique is advantageous over either marker used alone, in diagnosing sclerosing breast lesions. Our results indicate that p63 alone is not a useful myoepithelial cell marker if applied in large sclerosing breast lesions, however, in smaller lesions it is still of high value. On the contrary, SMA proved significantly useful in the evaluation of myoepithelial cells in larger but not in smaller complex sclerosing lesions. The myoepithelial cell cocktail has a staining sensitivity identical to that of SMA. Nevertheless, in a certain number of cases the cocktail might be useful in differentiating myoepithelial cells from stromal myofibroblasts or vascular smooth muscle cells due to the false impression of a higher staining intensity of the cocktail resulting from the expression of both nuclear and cytoplasmic/membranous antibodies that occupy a wider area of the cell under control.

HSP-70, C-myc and HLA-DR expression in patients with cutaneous malignant melanoma metastatic in lymph nodes.

HSP-70, C-myc and HLA-DR were examined in patients with cutaneous malignant melanoma metastatic to lymph nodes. Lymph-nodal fine-needle aspiration biopsies (FNABs) were analyzed and the results were correlated to other variables, such as the gender of the patients, Clark level and Breslow thickness of the primary tumor. Thirty cases of metastatic melanoma in lymph nodes from 30 patients with cutaneous malignant melanoma were studied. All patients (100%) had microscopic regional nodal metastasis and a recurrence of the lesion during the first two years. The HSP-70, C-myc and HLA-DR expressions were investigated immunocytologically, using the APAAP (alkaline phosphatase) method on the FNAB samples. The immunocytochemical expressions of HSP-70 protein, C-myc oncogene, and HLA-DR antigen were found in 18 cases (60%), in 14 cases (43.3%) and in 12 cases (40%), respectively. Clark levels were significantly associated with HSP-70 protein (< 0.01), C-myc oncogene expression (< 0.05) and HLA-DR antigen (< 0.01) expression. The HLA-DR antigen was also found to be related (< 0.05) to higher Breslow thickness (> 1.5 mm). The clinical course of malignant cutaneous melanoma is related to the expression of these indices, which seem to play a significant role in the metastasis and prognosis of this aggressive tumor. The immunocytochemical expression of HSP-70 in the malignant melanoma tumor could be of particular value in the identification of patients with poor prognosis.

Modulation of vascular endothelium by imatinib: a study on the EA.hy 926 endothelial cell line.

In this study the EA.hy 926 endothelial cell line--simulating endothelial cells--was treated with imatinib in order to define a possible anti-angiogenic role for imatinib. Dose and time response experiments were performed. Cell morphology was studied, while migration efficiency, intercellular permeability and VE-cadherin expression were assayed, both in the presence and in the absence of imatinib. Imatinib-induced EA.hy 926 cell apoptosis was also examined. Results showed that imatinib reduced the endothelial cell population, changed cell monolayer morphology and reduced cell-to-cell cohesiveness. Migration efficiency was significantly decreased while intercellular permeability was 2.76-fold increased in the presence of imatinib. Indirect immunofluorescence microscopy showed nearly complete down-regulation of VE cadherin in imatinib-treated cells. Furthermore, apoptotic activity was detected in imatinib-treated cells. Altogether our results support an antiangiogenic profile for imatinib that possibly contributes to its therapeutic potential.