The status of epidermal growth factor receptor in borderline ovarian tumours.

The majority of borderline ovarian tumours (BOTs) behave in a benign fashion, but some may show aggressive behavior. The reason behind this has not been elucidated. The epidermal growth factor receptor (EGFR) is known to contribute to cell survival signals as well as metastatic potential of some tumours. EGFR expression and gene status have not been thoroughly investigated in BOTs as it has in ovarian carcinomas. In this study we explore protein expression as well as gene mutations and amplifications of EGFR in BOTs in comparison to a subset of other epithelial ovarian tumours. We studied 85 tumours, including 61 BOTs, 10 low grade serous carcinomas (LGSCs), 9 high grade serous carcinomas (HGSCs) and 5 benign epithelial tumours. EGFR protein expression was studied using immunohistochemistry. Mutations were investigated by Sanger sequencing exons 18-21 of the tyrosine kinase domain of EGFR. Cases with comparatively higher protein expression were examined for gene amplification by chromogenic in situ hybridization. We also studied the tumours for KRAS and BRAF mutations. Immunohistochemistry results revealed both cytoplasmic and nuclear EGFR expression with variable degrees between tumours. The level of nuclear localization was relatively higher in BOTs and LGSCs as compared to HGSCs or benign tumours. The degree of nuclear expression of BOTs showed no significant difference from that in LGSCs (mean ranks 36.48, 33.05, respectively, p=0.625), but was significantly higher than in HGSCs (mean ranks: 38.88, 12.61 respectively, p< 0.001) and benign tumours (mean ranks: 35.18, 13.00 respectively, p= 0.010). Cytoplasmic expression level was higher in LGSCs. No EGFR gene mutations or amplification were identified, yet different polymorphisms were detected. Five different types of point mutations in the KRAS gene and the V600E BRAF mutation were detected exclusively in BOTs and LGSCs. Our study reports for the first time nuclear localization of EGFR in BOTs. The nuclear localization similarities between BOTs and LGSCs and not HGSCs support the hypothesis suggesting evolution of LGSCs from BOTs. We also confirm that EGFR mutations and amplifications are not molecular events in the pathogenesis of BOTs.

Nonenriched PCR Versus Mutant-Enriched PCR in Detecting Selected Epidermal Growth Factor Receptor Gene Mutations Among Nonsmall-Cell Lung Cancer Patients.

BACKGROUND: Biopsies obtained from lung cancers contain a mixture of cancerous and healthy tissues. The mutant-enriched polymerase chain reaction (ME-PCR) identifies low-level somatic DNA mutations within an excess wild-type sample. AIMS: This study aimed at comparing nonenriched PCR (NE-PCR) versus ME-PCR for the detection of two epidermal growth factor receptor (EGFR) gene mutations among nonsmall cell lung cancer patients. METHODS: Fifty lung tissue biopsies were screened for inframe TTAA deletions in exon-19 and the L858R point mutation in exon-21, using ME-PCR and NE-PCR, followed by capillary electrophoresis. RESULTS: Only exon-19 deletions were detected in 22% and 18% of cases using ME-PCR and NE-PCR, respectively. Diagnostic performance of the NE-PCR versus the ME-PCR serving as a "gold standard" revealed a sensitivity of 82%, and a specificity of 100%, with positive and negative predictive values of 100% and 95%, respectively, and an overall accuracy of 96%. Despite a strong agreement shown between the two assays (K=0.875), the NE-PCR showed an 18% false-negative rate in bronchoscopically obtained biopsies compared to ME-PCR. CONCLUSION: The false negativity encountered with NE-PCR in bronchoscopically obtained samples makes ME-PCR the technique of choice in such situations.

A novel gene, FGA7, is fused to RUNX1/AML1 in a t(4;21)(q28;q22) in a patient with T-cell acute lymphoblastic leukemia.

AML1 is among the most frequent targets of chromosomal rearrangements in human leukemias. We report here the molecular analysis of a t(4;21)(q28;q22) that has disrupted AML1 in a patient with de novo T-cell acute lymphoblastic leukemia. By using 3'-RACE analysis, we show that this rearrangement results in the fusion of a novel gene immediately downstream of exon 5 or exon 6 of AML1, indicating that the AML1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. The sequence of the novel gene, located at 4q28, does not have any significant homology with any of the known genes in the human GenBank DNA database. However, the first 118 bases are identical to a part of a human ovarian EST. Also, its high homology with mouse and rat sequences suggests that this sequence most probably represents a part of a novel gene, which we named FGA7 (Fused Gene 7 to AML1). Following the AML1 open reading frame, the FGA7 sequence encodes an unknown protein of 27 amino acids. We isolated three bacterial artificial chromosome (BAC) clones that contain the FGA7 sequence and confirmed the breakpoint of the gene on the patient's metaphase spreads by fluorescence in situ hybridization using these BACs as probes. RT-PCR and Northern blot analyses revealed that FGA7 is expressed in ovarian and skeletal muscle tissues. The predicted AML1-FGA7 chimeric proteins contained a limited number of residues fused to AML1 in a situation similar to that reported for the AML1-EAP fusion that is a product of t(3;21). It is possible that the expression of a constitutively shortened AML1 could compete with full-length AML1 and act as a dominant negative inhibitor of the promoters that the core binding factor activates.

Maternal serum alpha fetoprotein among pregnant females in Alexandria.

UNLABELLED: Maternal serum alpha fetoprotein (MSAFP) was introduced as a screening test for congenital malformations especially neural tube defects (NTDs) two decades ago. However, many factors were known to affect its level. From these are racial differences and maternal weight. The aim of the present work is to illustrate the normal distribution of MSAFP among working pregnant women in Alexandria in gestational age 16-18 weeks, to identify some of its determinants, and to determine the specificity and sensitivity of MSAFP for the detection of congenital anomalies and adverse pregnancy outcome. MATERIAL AND METHODS: A sample of 608 pregnant working women who were 16-18 week gestation was recruited for the study from the antenatal clinic affiliated to Gamal Abdel Nasser Health Insurance Hospital in Alexandria. The enrolled women were interviewed using a structured questionnaire and a blood sample was collected from each of them to measure the level of MSAFP. At the expected time of delivery, Gamal Abd el Nasser Health Insurance Hospital was visited to collect data about the outcome of pregnancy of the enrolled women. RESULTS: The median of MSAFP level for deliveries with no congenital anomalies were 25.5, 33.5, and 53.2 IU/ml, at gestational weeks 16, 17 and 18 respectively. The significant variables related positively to MSAFP level included abortion or stillbirth, congenital anomalies in the index pregnancy, gestational age, bleeding during pregnancy, gestational diabetes, twin pregnancy, consanguinity between maternal parents, history of congenital or genetic diseases in maternal family, and caesarian section deliveries. Fatigue score was negatively correlated to MSAFP level. Using MSAFP multiples of median (MOM), 42.9 % of abortions and stillbirths, 57.1 % of twin pregnancies, 31.25 % of preterm deliveries and 27.3 % of low birth weight had levels of 3 MOM or more. One fourth of the congenital anomalies were below 0.5 MOM and 41.7 % were at or above 3 MOM. The sensitivity of MSAFP test for the detection of NTDs (cutoff point 2.5+ MOM) or Down syndrome (cutoff point <0.5 MOM) among the study sample was 100% (CI: 19.8-100%). Specificity for NTDs was 92.7% (CI: 90.3-94.6%), while the specificity for Down syndrome was 89.1% (86.3-91.4%). The sensitivity for adverse pregnancy outcome (cutoff point <0.5 or 2.5+ MOM) was 41.6, and the specificity was 85.8%. In conclusion, the cutoff points of MSAFP of the study sample are different from those for other populations. Different factors affect the level of MSAFP including adverse pregnancy outcomes. It is recommended to introduce antenatal screening for congenital anomalies as a routine screening test during pregnancy using levels adapted from the local population for cutoff point determination.

A new translocation that rearranges the AML1 gene in a patient with T-cell acute lymphoblastic leukemia.

The AML1 gene (also known as RUNX1 or CBFA2), located in chromosome band 21q22, encodes a transcription factor which heterodimerizes with the CBFbeta protein forming a complex called human core binding factor (CBF). The CBF complex appears to regulate a number of genes important for hematopoiesis. AML1 is one of the most common targets of chromosomal rearrangements in human leukemias and has been involved in 14 chromosomal translocations to date. Here we report a new chromosomal translocation, t(4;21)(q31;q22) that disrupts the AML1 gene in a 12-year-old boy with newly diagnosed T-cell acute lymphoblastic leukemia (ALL). This is the first reported chromosomal translocation where AML1 is rearranged in childhood T-cell ALL. By metaphase fluorescence in situ hybridization analysis, the AML1 breakpoint was mapped using recombinant phage clones, and shown to be either immediately upstream or downstream of exon 5.