Molecular subtyping of breast cancer: Opportunities for new therapeutic approaches

Evidence is accumulating that breast cancer is not one disease but many separate diseases. DNA microarray-based gene expression profiling has demonstrated subtypes with distinct phenotypic features and clinical responses. Prominent among the new subtypes is 'basal-like' breast cancer, one of the 'intrinsic' subtypes defined by negativity for the estrogen, progesterone, and HER2/neu receptors and positivity for cytokeratins-5/6. Focusing on basal-like breast cancer, we discuss how molecular technologies provide new chemotherapy targets, optimising treatment whilst sparing patients from un-necessary toxicity. Clinical trials are needed that incorporate long-term follow-up of patients with well-characterised tumour markers. Whilst the absence of an obvious dominant oncogene driving basallike breast cancer and the lack of specific therapeutic agents are serious stumbling blocks, this review will highlight several promising therapeutic candidates currently under evaluation. Thus, new molecular technologies should provide a fundamental foundation for better understanding breast and other cancers which may be exploited to save lives.

Molecular subtyping of primary prostate cancer reveals specific and shared target genes of different ETS rearrangements

This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa), namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1) and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2) was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B) was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

Molecular subtyping of feline immunodeficiency virus from domestic cats in Australia

Objective To determine the prevalent subtypes of feline immunodeficiency virus (FIV) present in the domestic cat population of Australia. Method Blood samples were collected from 41 FIV antibody positive cats from four cities across Australia. Following DNA extraction, polymerase chain reaction (PCR) was performed to amplify the variable V3-V5 region of the envelope (env) gene. Genotypes were assessed by direct sequencing of PCR products and comparison with previously reported FIV sequences. Phylogenetic analysis allowed classification of the Australian sequences into the appropriate subtype. Results Of the 41 FIV samples, 40 were found to cluster with previously reported subtype A isolates, whilst the remaining sample grouped within subtype B. Conclusions Subtype A was found to be the predominant FIV subtype present in Australia, although subtype B was also found. These results broaden our knowledge of the genetic diversity of FIV and the associated implications for preventative, diagnostic and therapeutic approaches.

The phylogeny of Ureaplasma urealyticum based on the mba gene fragment

Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from an alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types which we have designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3, and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a, and 6b) thus facilitating high resolution typing of U. urealyticum. Inclusion of the reference strains for serovars 1, 3, 6, and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however this fragment was invariant within the parvo biovar. This study has shown that the sequence of the mba can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.

Reappraisal of immunohistochemical profiling of special histological types of breast carcinomas: a study of 121 cases of eight different subtypes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

First genetic identification of Cryptosporidium parvum subtype IIaA14G2R1in beef cattle in Brazil

The presence of Cryptosporidium spp. in a cattle herd registered with an outbreak of diarrhea was investigated and the the molecular subtyping of Cryptosporidium parvum was characterized. Fecal samples from 85 Nellore beef cattle (Bos indicus) were collected and examined with Ziehl-Neelsen modified staining method. Fifty-four cattle (63.52%) had Cryptosporidium spp. oocysts in their feces. Fragments of genes encoding the 18S ribosomal RNA subunit and a 60-kDa glycoprotein (gp60) were amplified by nested PCR accomplished in the 11 most heavily parasitized samples, and the amplicons were sequenced. Eight of the 11 analyzed samples were positive for 18S rRNA sequences and identified monospecific infections with C. parvum. Seven samples were positive for gp60 and identified subtypes IIaA15G2R1 (6/11) and IIaA14G2R1 (1/11). This report is the first for C. parvum subtype IIaA14G2R1 in beef cattle in Brazil.

Stratified analysis reveals chemokine-like factor (CKLF) as a potential prognostic marker in the MSI-immune consensus molecular subtype CMS1 of colorectal cancer

The Colorectal Cancer (CRC) Subtyping Consortium (CRCSC) recently published four consensus molecular subtypes (CMS’s) representing the underlying biology in CRC. The Microsatellite Instable (MSI) immune group, CMS1, has a favorable prognosis in early stage disease, but paradoxically has the worst prognosis following relapse, suggesting the presence of factors enabling neoplastic cells to circumvent this immune response. To identify the genes influencing subsequent poor prognosis in CMS1, we analyzed this subtype, centered on risk of relapse.
In a cohort of early stage colon cancer (n=460), we examined, in silico, changes in gene expression within the CMS1 subtype and demonstrated for the first time the favorable prognostic value of chemokine-like factor (CKLF) gene expression in the adjuvant disease setting [HR=0.18, CI=0.04-0.89]. In addition, using transcription profiles originating from cell sorted CRC tumors, we delineated the source of CKLF transcription within the colorectal tumor microenvironment to the leukocyte component of these tumors. Further to this, we confirmed that CKLF gene expression is confined to distinct immune subsets in whole blood samples and primary cell lines, highlighting CKLF as a potential immune cell-derived factor promoting tumor immune-surveillance of nascent neoplastic cells, particularly in CMS1 tumors. Building on the recently reported CRCSC data, we provide compelling evidence that leukocyte-infiltrate derived CKLF expression is a candidate biomarker of favorable prognosis, specifically in MSI-immune stage II/III disease.

Ocorrência e caracterização molecular de Cryptosporidium spp. em bezerros leiteiros da região noroeste do Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise molecular de estirpes de Escherichia coli enteropatogênica (EPEC) isoladas de animais e humanos

Pós-graduação em Microbiologia Agropecuária - FCAV