Validation of an oligo-gene signature for the prognostic stratification of ductal carcinoma in situ (DCIS).

Current evidence suggests that the majority of DCIS lesions do not progress to invasive carcinoma, and overtreatment of DCIS is a significant problem. We previously reported an 8-gene signature that differentiated microdissected low-grade (LG) DCIS lesions with and without associated stromal invasion, based on differential DNA copy number changes detected by quantitative (q) PCR. The current study was undertaken to validate our candidate breast cancer invasion gene panel in a larger series of non-microdissected LG DCIS cases, and to investigate its potential utility in intermediate-grade (IG) and high-grade (HG) DCIS. Representative paraffin blocks were selected from 267 resected DCIS cases with 5-15 years of follow-up (139 pure DCIS ["PD"] and 128 mixed DCIS with associated invasion ["MD"]). These included 171 LG, 46 IG and 50 HG DCIS cases. Gene copy number changes were determined by qPCR, and their differential distribution in the PD and MD subgroups was evaluated. As an alternate platform, we employed immunohistochemistry (IHC). Novel IHC assays were developed for all eight candidate genes, and increased or reduced protein expression was manually scored. Separate multi-gene models were developed for qPCR and IHC to distinguish progressing and non-progressing DCIS lesions. By qPCR analysis, a panel of six genes, as well as CELSR1 alone (a potential invasion suppressor), differentiated PD and MD cases in LG and IG, but not in HG DCIS. By IHC, a panel of three genes, as well as GRAP2 alone (a potential invasion promoter), also distinguished PD and MD cases in LG and IG, but not in HG DCIS. The combination of CELSR1 (by qPCR) and GRAP2 (by IHC) had the best discriminatory power (p = 0.00004). Assays testing either or both of these genes have the potential to become important adjuncts for choosing appropriate treatment for LG/IG DCIS patients.

Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

INTRODUCTION: Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)]. METHODS: Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy. RESULTS: TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells. CONCLUSION: These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

Chemotherapy enriches for an invasive triple-negative breast tumor cell subpopulation expressing a precursor form of N-cadherin on the cell surface.

BACKGROUND: Although most triple-negative breast cancer (TNBC) patients initially respond to chemotherapy, residual tumor cells frequently persist and drive recurrent tumor growth. Previous studies from our laboratory and others' indicate that TNBC is heterogeneous, being composed of chemo-sensitive and chemo-resistant tumor cell subpopulations. In the current work, we studied the invasive behaviors of chemo-resistant TNBC, and sought to identify markers of invasion in chemo-residual TNBC. METHODS: The invasive behavior of TNBC tumor cells surviving short-term chemotherapy treatment in vitro was studied using transwell invasion assays and an experimental metastasis model. mRNA expression levels of neural cadherin (N-cadherin), an adhesion molecule that promotes invasion, was assessed by PCR. Expression of N-cadherin and its precursor form (pro-N-cadherin) was assessed by immunoblotting and flow cytometry. Pro-N-cadherin immunohistochemistry was performed on tumors obtained from patients pre- and post- neoadjuvant chemotherapy treatment. RESULTS: TNBC cells surviving short-term chemotherapy treatment exhibited increased invasive behavior and capacity to colonize metastatic sites compared to untreated tumor cells. The invasive behavior of chemo-resistant cells was associated with their increased cell surface expression of precursor N-cadherin (pro-N-cadherin). An antibody specific for the precursor domain of N-cadherin inhibited invasion of chemo-resistant TNBC cells. To begin to validate our findings in humans, we showed that the percent cell surface pro-N-cadherin (+) tumor cells increased in patients post- chemotherapy treatment. CONCLUSIONS: TNBC cells surviving short-term chemotherapy treatment are more invasive than bulk tumor cells. Cell surface pro-N-cadherin expression is associated with the invasive and chemo-resistant behaviors of this tumor cell subset. Our findings indicate the importance of future studies determining the value of cell surface pro-N-cadherin as: 1) a biomarker for TNBC recurrence and 2) a therapeutic target for eliminating chemo-residual disease.

Prostate-derived Ets factor is overexpressed in serous epithelial ovarian tumors.

The frequent overexpression of prostate-derived Ets factor (PDEF) mRNA in ovarian cancer has been previously reported. The aim of this study was to evaluate PDEF protein expression in ovarian cancer and how this expression might vary at different stages of epithelial ovarian tumors in comparison to normal ovary. A new rabbit polyclonal antibody to PDEF was prepared, and immunohistochemistry was performed on tissue sections from 12 normal ovaries, 10 cases of benign serous cystadenoma, 17 cases of low malignant potential tumor, 19 cases of stage 1, and 15 cases of advanced stage primary epithelial (serous) ovarian carcinomas and their peritoneal metastases. Expression levels were assessed based on the percentage of positively staining cells and the intensity of staining. All 12 normal ovary and 10 benign serous cystadenoma cases were negative for PDEF expression. In contrast, 6 of 17 (35%) low malignant potential tumors, 5 of 19 (27%) stage 1, and 5 of 15 (33%) advanced stage ovarian tumors stained positive for PDEF expression. Together, these results show frequent overexpression of PDEF protein in epithelial ovarian tumors and its lack of expression in normal ovary and cystadenomas, and this supports a role for PDEF in ovarian tumorigenesis. Furthermore, these results suggest that PDEF is a potential marker and target in ovarian cancer.

Cyclooxygenase-2 (COX-2) levels before and after chemotherapy: a study in rectal cancer.

OBJECTIVES: Induction of cyclooxygenase-2 (COX-2) by inflammatory mediators, oncogenes, and carcinogens has been demonstrated in preclinical models. However, there are limited clinical data regarding COX-2 induction by chemotherapy or radiation. Experimental data suggest cross-talk between the EGFR and COX-2 pathways. The aim of this study was to analyze the expression of COX-2 before and after chemoradiation (CRT) and correlate the same with tumor (T) down-staging and survival. Similar data were obtained for EGFR expression before and after chemoradiation. METHODS: Archival paraffin-embedded tumor specimens from patients undergoing CRT between 1995 and 2001 were analyzed. COX-2 expression was measured by immunohistochemistry (IHC), using the 160112 COX-2 mouse monoclonal antibody. For EGFR, we used mouse monoclonal Ab-10. Standard immunoperoxidase technique was used to detect the avidin- biotin peroxidase complex. Staining in tumor tissue was visually scored and confirmed by an image analyzer (ACIS; ChromaVision Medical Systems, Inc, San Juan Capistrano, CA). RESULTS: Twenty pretreatment biopsy samples from rectal cancer patients and their paired, post-CRT surgical specimens (n = 17) were analyzed. Three cases had no primary tumor after CRT. COX-2 expression was noted in 19 of 20 pretreatment samples and 17 of 17 surgical specimens. EGFR expression was noted in 10 cases pretreatment. Six patients with weakly positive COX-2 expression pretreatment had increased COX-2 expression after CRT, whereas in 1 patient the expression decreased after CRT. No EGFR induction was noted. There was no statistical association between EGFR and COX-2 expression in this data set. Median survival for the entire cohort was 38.9 months. There was no difference in survival between the COX-2 induced and noninduced groups. CONCLUSIONS: COX-2 induction was seen with CRT in this population of rectal cancer patients. Prognostic significance of this induction remains to be defined in a larger cohort.

PHYLOGEOGRAPHY OF SPOTTED OWL (STRIX OCCIDENTALIS) POPULATIONS BASED ON MITOCHONDRIAL DNA SEQUENCES: GENE FLOW, GENETIC STRUCTURE, AND A NOVEL BIOGEOGRAPHIC PATTERN.

Mitochondrial DNA control region sequences of spotted owls (Strix occidentalis) allowed us to investigate gene flow, genetic structure, and biogeographic relationships among these forest-dwelling birds of western North America Estimates of gene flow based on genetic partitioning and the phylogeography of haplotypes indicate substantial dispersal within three long-recognized subspecies. However, patterns of individual phyletic relationships indicate a historical absence of gene flow among the subspecies, which are essentially monophyletic. The pattern of haplotype coalescence enabled us to identify the approximate timing and direction of a recent episode of gene flow from the Sierra Nevada to the northern coastal ranges. The three subspecies comprise phylogenetic species, and the northern spotted owl (S. o. caurina) is sister to a clade of California (S. o. occidentalis) plus Mexican spotted owls (S o lucida); this represents a novel biogeographic pattern within birds. The California spotted owl had substantially lower nucleotide diversity than the other two subspecies; this result is inconsistent with present patterns of population density A causal explanation requires postulating a severe bottleneck or a selective sweep, either of which was confined to only one geographic region.