Disclosed are new methods comprising the use of in situ hybridization to detect abnormal nucleic acid sequence copy numbers in one or more genomes wherein repetitive sequences that bind to multiple loci in a reference chromosome spread are either substantially removed and/or their hybridization signals suppressed. The invention termed Comparative Genomic Hybridization (CGH) provides for methods of determining the relative number of copies of nucleic acid sequences in one or more subject genomes or portions thereof (for example, a tumor cell) as a function of the location of those sequences in a reference genome (for example, a normal human genome). The intensity(ies) of the signals from each labeled subject nucleic acid and/or the differences in the ratios between different signals from the labeled subject nucleic acid sequences are compared to determine the relative copy numbers of the nucleic acid sequences in the one or more subject genomes as a function of position along the reference chromosome spread. Amplifications, duplications and/or deletions in the subject genome(s) can be detected. Also provided is a method of determining the absolute copy numbers of substantially all RNA or DNA sequences in subject cell(s) or cell population(s).

Comparative genomic hybridization (cgh)

Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

/pdf/distinctive-serum-protein-profiles-involving-abundant-14lob0qahz.pdf

Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan system Two fluorogenic probes anneal to the target between primers for c-erbB-2 and β-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine) During the extension phase of the PCR cycle, the 5'→3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 094, P <001) This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens

/pdf/quantitative-polymerase-chain-reaction-based-homogeneous-68551dyc5v.pdf

Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification.

The first part of this two-part review of established and emerging breast cancer biomarkers and molecular diagnostics considers breast cancer predisposition, screening tests for diagnosis, diagnosis using small specimens and metastatic lesions, micrometastatic disease and breast cancer prognosis assessment. Prognostic factors covered in this review include: cytogenetic markers, DNA ploidy and S phase determination, cell proliferation markers, cell cycle regulators and growth factor measurements including epithelial growth factor receptor, HER-2/neu and a variety of other relevant molecules controlling proliferation, differentiation and angiogenesis. The first section of part two will continue the consideration of breast cancer prognostic factors including oncogenes, tumor suppressor genes, cell adhesion molecules, invasion-associated proteins and proteases, hormone receptor proteins, drug resistance proteins, apoptosis regulators, transcription factors, telomerase, DNA repair and methylation and transcrip...

/pdf/breast-cancer-biomarkers-and-molecular-medicine-74wxg0dyd4.pdf

Breast cancer biomarkers and molecular medicine.

Flavonoid-rich extracts from the mature roots of Scutellaria baicalensis have been shown to exhibit antiproliferative effects on various cancer cell lines. We assessed the ability of an ethanolic extract of S. baicalensis root to inhibit the proliferation of malignant glioma cells. Cell lines derived from primary and recurrent brain tumors from the same patient and cells selected for resistance to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) were used to identify antiproliferative effects of this extract when used alone and in conjunction with BCNU. Results indicated that Scutellaria baicalensis not only inhibits cellular growth in recurrent and drug resistant brain tumor cell lines, but also demonstrates an increased inhibitory effect when used in conjunction with BCNU. The results of this study support the efficacy of S. baicalensis as an anticancer agent for glioblastomas multiforme and a potential adjuvant treatment to current chemotherapeutic agents used in the treatment of both primary and recurrent GBMs. Further studies of the effects of individual flavonoids alone and in combination with each other and with currently used therapies are needed.

/pdf/anticancer-activity-of-extracts-derived-from-the-mature-bbk6j2hnvv.pdf

Anticancer activity of extracts derived from the mature roots of Scutellaria baicalensis on human malignant brain tumor cells.

Background. Identifying markers that have the potential to predict tumor behavior is important in breast cancer because of the variability in clinical disease progression. Genetic alterations in tumors may appear as changes in total DNA content, individual chromosomes, single genes, or gene expression. Alteratian in DNA content is an imprecise but accessible measurement of the genome. Diploid tumors have been associated with a better clinical outcame, and increased ploidy correlates with ather indicators of poor prognosis. Concurrent analysis of DNA content with markers of genetic expression is feasible (e.g., myc oncogene) and may increase its prognostic power

Genetic markers as prognostic indicators in breast cancer.

Background. Although some patients with malignant gliomas respond to treatment with chemotherapeutic agents like BCNU, tumor recurrence inevitably occurs, heralding the development of chemoresistance. Treating and/or preventing chemoresistance requires distinguishing newly developed resistance from the presence of intrinsically resistant cells in the primary tumor population. This study relates the chromosomal complements of freshly resected astrocytomas to the cells' chemosensitivity and ultimately to the patients' response to treatment.



Methods. The authors dissociated 31 freshly resected human gliomas (5 astrocytomas, 10 anaplastic astrocytomas, 16 glioblastomas multiforme) into single cells, and performed cytogenetic analysis and BCNU sensitivity testing using the colony-forming assay (CFA) on first division cells from these tumors.



Results. The major cytogenetic abnormalities involved the loss of a sex chromosome in all three classes of gliomas and the gain of chromosome 7 in anaplastic astrocytoma and glioblastoma multiforme; clonal marker chromosomes were observed in only anaplastic astrocytoma and glioblastoma multiforme with no common rearrangement observed among the tumors. The five astrocytomas were near-diploid (2n°, 35–57 chromosomes/cell), and all were resistant to BCNU. Seven of ten anaplastic astrocytomas were composed primarily of 2n° cells and were BCNU resistant. Three other anaplastic astrocytomas had a 39% or greater representation of 4n° cells (88–101 chromosomes/cell), and these tumors were sensitive to BCNU. Ten of 16 glioblastomas multiforme were composed predominantly of 2n° cells and were resistant to carmustine. Six other glioblastomas multiforme had at least 41% 3n° (58–87 chromosomes/metaphase) and 4n° cell populations and were sensitive to carmustine. Thus, gliomas demonstrating BCNU sensitivity were more than 60% hyperdiploid (60 or more chromosomes/metaphase) with 1 to 8 clonal marker chromosomes and multiple clonal populations involving complex karyotypic deviations. In contrast, all 22 resistant tumors were composed primarily of near-diploid cells. Only 4 of 22 tumors had a clonal marker, and the chromosome ploidy changes were less extensive.



Conclusions. In freshly resected untreated human gliomas, BCNU is most effective against hyperdiploid cells that have extensive ploidy changes and chromosome rearrangement, whereas resistance to carmustine is characteristic of near-diploid populations with few ploidy changes and rearranged chromosomes. This observation was consistent for all three classes of gliomas.

Chromosome number and carmustine sensitivity in human gliomas

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Genetic markers as prognostic indicators in breast cancer.

Chromosome number and carmustine sensitivity in human gliomas