The role of the urokinase family in invasion by breast cancer

Breast cancer is the leading cause of cancer mortality in women in the Western world. Its growth and development is directly governed by endogenous steroids and growth factors. Endocrine therapy is the most effective form of treatment for a proportion of breast cancer patients. Work from several laboratories has shown that breast cancer cells metastasise via a pathway that involves the plasminogen activator system and metalloproteinases. Thus, the plasminogen activator system, which consists of urokinase (uPA), its inhibitor (PAI-1) and receptor (uPAR), is central to the process of invasion and metastasis. Given their important role, it is hardly surprising that these three proteins have been used as biological markers of patient prognosis. uPA and PAI-1 has been significantly implicated as markers of aggressive breast cancer. The first step of invasion involves the conversion of plasminogen into plasmin by uPA. uPA is localised on the cell surface (bound to its receptor uPAR) and is principally regulated by two inhibitors, plasminogen activators type-1 and type-2 (PAI-1 and PAI-2). Possessing a wide substrate specificity, plasmin in turn activates a variety of enzymes which degrade different components of the extracellular matrix such as laminin, fibronectin, collagenase(s), proteoglycans etc. The present study was conducted to further understand the effect(s) of different growth factors and steroid hormones on the proliferation and invasive potential of two well characterised human breast cancer cell lines, MCF-7 (hormone sensitive) and MDA-MB-231 (hormone insensitive). Furthermore, extracellular matrix (ECM) was used to determine the effect, if any that support substrate might have on the levels of plasminogen activators and growth rate. Plasminogen activators were quantified both at the mRNA and protein levels, using RT-PCR and ELISA, respectively. uPA activity was determined by chromogenic assay and the invasive properties of the two cell lines were determined using the Boyden invasion chamber. In the initial phase, proliferation of each cell line on both substrata, plastic and EHS, was studied after treatment with growth factors (TGFalpha, TGFbeta and EGF), steroid hormones (E2 and Pg) and anti-oestrogen, tamoxifen. Different amounts of each growth factor and hormone were used and cell numbers were obtained after 24, 48 and 72 hours. These studies showed that growth of MCF-7 cells was stimulated significantly by exogenous TGFalpha and EGF, on both EHS and plastic with cells being more sensitive to growth factors on EHS. Proliferation of MDA cells was only slightly increased by the same growth factors on either substrate. By contrast, TGFbeta inhibited the growth of MDA cells, on both surfaces. However, the concentrations required for inhibition on the two substrata were enormously different. E2, Pg, and TAM (an anti-oestrogen) also stimulated the growth of MCF-7 cells on both plastic and EHS. For quantification of uPA, uPAR and PAI-1, a combination of Western blotting and ELISA, using specific monoclonal antibodies, was employed to monitor the relative amounts of these proteins after treating the cell lines with growth factors and hormones. These studies showed that the MDA cell line expressed uPA and PAI-1 proteins whereas uPA was hardly delectable in the MCF-7 cell line. Moreover, qualitative RT-PCR analysis clearly showed that uPAR and PAI-1 transcripts were present in both MDA and MCF-7 cells. These results clearly show that various growth factors and/or hormones not only have an effect on the proliferative properties of cell lines but they also change the relative amounts of uPA, PAI-1 and uPAR and that these changes can be reflected in invasive activity. The ECM contributes to the overall response of these cells. (Abstract shortened by ProQuest.)

Utilisation Patterns and Treatment Outcomes of EGFR-Tyrosine Kinase Inhibitors in EGFR-mutant Advanced Lung Carcinoma in the Pakistani-Asian Population: A Real-world Data Study

Introduction: Data on the utilisation of epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitors (TKIs) and their clinical outcomes in a heterogeneous Pakistani-Asian population have not been previously reported. This manuscript presents the first account of the clinical outcomes of EFGR-TKIs in EGFR-mutant lung adenocarcinoma among Pakistani- Asians. Materials and Methods: A real-world data study was conducted on all advanced lung cancer patients harbouring EGFR-mutations from the cancer registry of Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan. We identified three different patterns of the use of EGFR-TKIs (Groups 1, 2 and 3) that reflect the ground realities of cancer care and delivery in Pakistan. We also noted a significant proportion of patients (Group 4) without access to EGFR TKIs. We compared the objective response rates (ORR), progression-free survival (PFS) and overall survival (OS) of each of the four groups and reported their toxicity profile. Results: Within the limitations of a retrospective analysis, we saw differences in the frequency of EGFR mutations in this population. However, response rates and long-term outcomes of EGFR TKI therapy were comparable with the existing data. The overall use of EGFR TKIs led to a superior outcome in ORR, PFS and OS compared to chemotherapy alone; (77.8% vs. 50.0%, 16.3 vs. 10.7 months; P = 0.099; 85.6 vs. 25.9 months, respectively; P = 0.13). Conclusion: Except for modest differences, EGFR-mutant advanced lung adenocarcinoma outcomes among Pakistani-Asians are comparable to those of other populations

Down-regulation of DNMT3b in PC3 cells effects locus-specific DNA methylation, and represses cellular growth and migration

<p>Abstract</p> <p>Background</p> <p>Aberrations in DNA methylation patterns promote changes in gene expression patterns and are invariably associated with neoplasia. DNA methylation is carried out and maintained by several DNA methyltransferases (DNMTs) among which DNMT1 functions as a maintenance methylase while DNMT3a and 3b serve as de novo enzymes. Although DNMT3b has been shown to preferentially target the methylation of DNA sequences residing in pericentric heterochromatin whether it is involved in gene specific methylation remains an open question. To address this issue, we have silenced the expression of DNMT3b in the prostate-derived PC3 cells through RNA interference and subsequently studied the accompanied cellular changes as well as the expression profiles of selected genes.</p> <p>Results</p> <p>Our results demonstrate that DNMT3b depletion results in increased apoptosis and reduced migration of PC3 cells compared to the untransfected control cells. Reduced DNMT3b expression resulted in hypomethylation of retinoblastoma (Rb), retinoic-acid receptor β (RAR-β), and adenomatous polyposis coli (APC) gene promoters, and also culminated in increased expression of <it>CDKN3 </it>and <it>cytochrome b5</it>. Although DNMT3b silenced cells were found to have reduced growth and migratory potential, there was no apparent changes in their invasive ability compared to the parental PC3 cell line.</p> <p>Conclusion</p> <p>Our findings reveal that DNMT3b preferentially targets certain gene promoters in PC3 cells and that its depletion significantly reduces growth and migration of PC3 cells.</p

Silencing of MBD1 and MeCP2 in prostate-cancer-derived PC3 cells produces differential gene expression profiles and cellular phenotypes

Alterations in genomic CpG methylation patterns have been found to be associated with cell transformation and neoplasia. Although it is recognized that methylation of CpG residues negatively regulates gene expression, how the various MBPs (methyl-binding proteins) contribute to this process remains elusive. To determine whether the two well characterized proteins MeCP2 (methyl-CpG-binding protein 2) and MBD1 (methyl-CpG-binding domain 1) have distinct or redundant functions, we employed RNAi (RNA interference) to silence their expression in the prostate cancer-derived PC3cell line, and subsequently compared cell growth, invasion and migration properties of these cell lines in addition to their respective mRNA-expressionprofiles. Cells devoid of MeCP2 proliferated more poorly compared with MBD1-deficient cells and the parental PC3 cells. Enhanced apoptosis was observed in MeCP2-deficient cells, whereas apoptosis in parental and MBD1-deficient cells appeared to be equivalent. Boyden chamber invasion and wound-healing migration assays showed that MBD1-silenced cells were both more invasive and migratory compared with MeCP2-silenced cells. Finally, gene chip microarray analyses showed striking differences in the mRNA-expression profiles obtained from MeCP2- and MBD1-depleted cellsrelative to each other as well as when compared with control cells. The results of the present study suggest that MeCP2 and MBD1 silencing appear to affect cellular processes independently in vivo and that discrete sets of genes involved in cellular proliferation, apoptosis, invasion and migration are targeted by each protein

Multi-center real-world comparison of the fully automated Idylla (TM) microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla (TM) MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla (TM) testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla (TM) results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla (TM) MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had >= 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla (TM) MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla (TM) MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla (TM) MSI Assay and routine tests.Peer reviewe

Translocation t(1;19) in acute precursor B-cell lymphoblastic Leukaemia in paediatric patients- Pakistani population

Objective: To determine the impact of translocation t(1;19) in paediatric patients diagnosed with precursor B-cell acute lymphoblastic leukaemia. Methods: The retrospective study was conducted at the Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan, and comprised data from January 2012 to January 2018 of paediatric patients diagnosed with precursor B-cell acute lymphoblastic leukaemia. Data of patients having t(1;19) translocation with or without complex karyotype formed group A, while data of patients without any cytogenetic abnormality formed the control group B. Relapse and event-free survival were calculated and the outcomes were compared between the groups. Data was analysed using SPSS 20. Results: Of the 450 subjects whose data was analysed, 84(18.7%) were included; 25(30%) in group A and 59(70%) were in group B. There were 21(84%) males and 4(16%) females in group A with mean age on presentation 3.68±0.6 years compared to 41(69.4%) males and 18(30.5%) females with mean age on presentation 4.0±0.92 (p>0.05). There were no differences between the groups in terms of baseline markers (p>0.05). The relapse and event-free survival rates were also not significantly different between the groups (p>0.05). Conclusion: There was no significant difference related to outcomes of precursor B-cell acute lymphoblastic leukaemia patients having t(1;19) translocation with or without complex karyotype and those without any cytogenetic abnormality, indicating that translocation t(1;19)-positive patients do not need treatment intensification. Key Words: Acute Pre-B ALL, Children, t (1;19), Oncoprotein E2A-PBX1

Disease Spectrum in COVID-19 Cohort with Travel History from Iran

Background: Coronavirus disease 2019 (Covid-19), declared as a pandemic in March 2020, is an acute respiratory tract illness caused by coronavirus 2 (SARS-CoV2) with clinical manifestations ranging from mild upper respiratory tract symptoms to severe pneumonia. Objectives: To determine the disease spectrum of Covid-19 in a cohort with a travel history from Iran. Materials & Methods: This cross-sectional study with a retrospective collection of data was conducted at Agha Khan University, Karachi from 15th March to 19th April 2020. One hundred and fifty-five laboratory-confirmed cases of Covid-19 were recruited from a government quarantine facility. Data were obtained from the Punjab Emergency Services (Rescue 1122) database where a record of SARS-CoV-2 positive cases and quarantined persons is maintained. Study subjects with a travel history to Iran were contacted by telephone to obtain information about demographics, symptoms, and co-morbid conditions.  SPSS version 24 was used to analyze the data. Frequencies and percentages were calculated. Results: Among the returning travelers, 213 had laboratory-confirmed Covid-19, out of which 155 were included in this study. 56.1% were males with a mean age of 40 years. Among the study participants, 91.6% remained asymptomatic throughout the stay, while 8.4 % became symptomatic. 77.5% of the participants had received BCG vaccination in childhood. Among symptomatic cases 15.4% had asthma and 7.7% had hypertension. The most common clinical manifestation was cough which was present in 38.5% of the study participants. None died among the study participants. Conclusion: A mild presentation of COVID-19 was seen in our study participants with 91.6% among them being asymptomatic, while 8.4% were symptomatic. There was a high positivity rate in males as compared to females.

Population-based surveillance for severe rotavirus gastroenteritis in children in Karachi, Pakistan

The incidence of rotavirus-associated severe diarrhoea and distribution of rotavirus genotypes in children less than five years of age was determined in two low-income communities in Karachi, Pakistan. Over a two-year period, 717 children met eligibility criteria for severe diarrhoea and stools were obtained from 575 (80%) with 97 (17%) being rotavirus positive. Adjusted annual rates of severe rotavirus diarrhoea in children less than five years and less than one year were respectively 5.7 and 16.9 per 1000 in community A, and 8.1 and 25.4 per 1000 child years of observation in community B. An estimated 1 in 40 infants experience a severe episode of rotavirus gastroenteritis annually in Pakistan. The most common rotavirus strains were G9P[8] (15%), G1P[8] (13%) and G1[P4] (8.4%). This information will inform policy decisions about the introduction of rotavirus vaccines