Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures.

Our goal was to establish primary cultures from dissociation of breast tumors in order to provide cellular models that may better recapitulate breast cancer pathogenesis and the metastatic process. Here, we report the characterization of six cellular models derived from the dissociation of primary breast tumor specimens, referred to as "dissociated tumor (DT) cells." In vitro, DT cells were characterized by proliferation assays, colony formation assays, protein, and gene expression profiling, including PAM50 predictor analysis. In vivo, tumorigenic and metastatic potential of DT cultures was assessed in NOD/SCID and NSG mice. These cellular models differ from recently developed patient-derived xenograft models in that they can be used for both in vitro and in vivo studies. PAM50 predictor analysis showed DT cultures similar to their paired primary tumor and as belonging to the basal and Her2-enriched subtypes. In vivo, three DT cultures are tumorigenic in NOD/SCID and NSG mice, and one of these is metastatic to lymph nodes and lung after orthotopic inoculation into the mammary fat pad, without excision of the primary tumor. Three DT cultures comprised of cancer-associated fibroblasts (CAFs) were isolated from luminal A, Her2-enriched, and basal primary tumors. Among the DT cells are those that are tumorigenic and metastatic in immunosuppressed mice, offering novel cellular models of ER-negative breast cancer subtypes. A group of CAFs provide tumor subtype-specific components of the tumor microenvironment (TME). Altogether, these DT cultures provide closer-to-primary cellular models for the study of breast cancer pathogenesis, metastasis, and TME.

ER re-expression and re-sensitization to endocrine therapies in ER-negative breast cancers.

Breast cancer is the leading cause of cancer amongst women in the westernized world. The presence or absence of ERalpha in breast cancers is an important prognostic indicator. About 30-40% of breast cancers lack detectable ERalpha protein. ERalpha- breast cancers are resistant to endocrine therapies and have a worse prognosis than ERalpha+ breast cancers. Since expression of ERalpha is necessary for response to endocrine therapies, investigational studies are ongoing in order to understand the generation of the ERalpha- phenotype and develop interventions to restore ERalpha expression in ERalpha- breast cancers. DNA methylation and chromatin remodeling are two epigenetic mechanisms that have been linked with the lack of ERalpha expression and in these cases; demethylation of the ERalpha promoter or treatment with HDAC inhibitors shows promise in restoring ERalpha expression in ERalpha- breast cancers. Two additional potential mechanisms underlying generation of the ERalpha- phenotype involve E6-AP and Src, both of which have been shown to be elevated in ERalpha- breast cancer and can drive the proteasomal degradation of ERalpha. Recently, studies have demonstrated that upregulated growth factor signaling due to hyperactive MAPK activity significantly contributes to generation of the ERalpha- phenotype and that inhibition of MAPK activity can cause re-expression of the ERalpha and restore sensitivity to endocrine therapies. Given the challenges in treating ERalpha- breast cancer, understanding and manipulating the cellular mechanisms that effect expression of ERalpha are imperative in order to restore sensitivity to endocrine therapies and to design novel therapeutics for the treatment of ERalpha- breast cancers.

Therapeutic vaccination for HPV induced cervical cancers.

Cervical Cancer is the second leading cause of cancer-related deaths in women worldwide and is associated with Human Papillomavirus (HPV) infection, creating a unique opportunity to treat cervical cancer through anti-viral vaccination. Although a prophylactic vaccine may be available within a year, millions of women, already infected, will continue to suffer from HPV-related disease, emphasizing the need to develop therapeutic vaccination strategies. A majority of clinical trials examining therapeutic vaccination have shown limited efficacy due to examining patients with more advanced-stage cancer who tend to have decreased immune function. Current trends in clinical trials with therapeutic agents examine patients with pre-invasive lesions in order to prevent invasive cervical cancer. However, longer follow-up is necessary to correlate immune responses to lesion regression. Meanwhile, preclinical studies in this field include further exploration of peptide or protein vaccination, and the delivery of HPV antigens in DNA-based vaccines or in viral vectors. As long as pre-clinical studies continue to advance, the prospect of therapeutic vaccination to treat existing lesions seem good in the near future. Positive consequences of therapeutic vaccination would include less disfiguring treatment options and fewer instances of recurrent or progressive lesions leading to a reduction in cervical cancer incidence.

Optimization of PCR based detection of human papillomavirus DNA from urine specimens.

BACKGROUND: Human papillomavirus (HPV) causes cervical cancer. Current screening requires a yearly pelvic exam and Pap smear. However, these procedures are impractical for screening all women at risk for disease. Urine sampling has been successfully utilized to screen for Chlamydia trachomatis (CT) and Neisseria gonorrhoreae (NG) infections and has been considered for HPV DNA detection by several investigators. However, no study to date has been performed to specifically optimize HPV detection in urine. OBJECTIVES: To compare handling and extraction techniques in order to optimize the HPV specific PCR system in urine specimens. STUDY DESIGN: Examination of 10 characteristics that may contribute to PCR inhibition in urine was performed utilizing 10SG mulitstixs. Five different DNA extraction methods were compared in spiked specimens and in 10 clinical specimens. After the optimal extraction technique was identified, concentration of the sample with and without prior dilution was compared to the original protocol. Lastly, specimen handling was compared between immediate processing, refrigerating overnight, or freezing overnight. RESULTS AND CONCLUSIONS: the presence of protein in urine enhanced amplification while nitrites decreased amplification. Of the extraction methods tested, the QIAamp DNA Mini Kit demonstrated the best amplification from urine samples spiked with HPV DNA and clinical specimens. The addition of a dilution step and a concentration step before applying the Qiagen protocol further increased amplification of beta-globin (from 50 to 63%) and the HPV L1 gene (from 13 to 33%). Lastly, refrigerating the specimens at 4 degrees C overnight appears to produce better amplification (62% beta-globin and 17% HPV positive) than either immediate processing (46% beta-globin and 13% HPV+) or freezing the specimen for 24h prior to processing (46% beta-globin and 10% HPV+). In these studies, amplification was low despite optimization. Additional improvements are required prior to clinical application of a urine-based HPV DNA detection system.

Peptide-based vaccines for cancer immunotherapy.

One approach in the immunotherapy of cancer patients involves vaccination with peptides derived from tumour-associated antigens specifically designed to associate with T cells in the context of major histocompatibility complex (MHC) class I or II molecules. Several clinical trials in different tumour types have been conducted utilising this vaccination strategy. The majority of trials indicate that peptide vaccination has few toxicities associated with its administration, but disparities exist between in vitro and clinical responses. However, this represents an evolving field and, thus, it is difficult to draw firm conclusions concerning the efficacy of peptide-based vaccines for cancer immunotherapy. Improvements to peptide vaccination, including the addition of various adjuvants, the utilisation of peptide-pulsed dendritic cells, multipeptide vaccinations, the addition of helper peptides and peptide delivery through the use of mini-genes, are encouraging and serve as important guides for future research.

The efficacy of a DNA vaccine containing inserted and replicated regions of the E7 gene for treatment of HPV-16 induced tumors.

A majority of cervical cancers are associated with Human Papillomavirus (HPV)-16. A DNA vaccine (E7IR) was designed for prophylactic and therapeutic treatment of HPV-16+ tumors containing two repeats of the E7 gene to inactivate transformation and duplicate available epitopes. Mice were vaccinated then tumor challenged, or challenged and then immunized and monitored for tumor volume and survival. Splenocytes were utilized for in vivo CTL assays. The E7IR vaccine demonstrated decreased tumor volume and enhanced survival in prophylactic and therapeutic experiments and improved CTL-mediated lysis. The E7IR vaccine shows promise in prevention of tumor formation and elimination of established tumors.

Distribution of human papillomavirus type 16 variants in human immunodeficiency virus type 1-positive and -negative women.

The prevalence of human papillomavirus type 16 E6 variant lineages was characterized in a cross-sectional study of 24 human immunodeficiency virus type 1 (HIV)-positive and 33 HIV-negative women in New Orleans. The European prototype was the predominant variant in the HIV-negative women (39.4 %), while in the HIV-positive women the European 350G variant was predominant (29.1 %). In exact logistic regression models, HIV-positive women were significantly more likely to harbour any variant with a nucleotide G-350 mutation compared with HIV-negative women [58.3 % vs 21.1 %; adjusted odds ratio (AOR)=6.28, 95 % confidence interval (CI)=1.19-46.54]. Models also revealed a trend towards increased prevalence of Asian-American lineage in HIV-positive women compared with HIV-negative women (25.0 % vs 6.0 %; AOR=6.35, 95 % CI=0.77-84.97). No association was observed between any variant and cytology or CD4 cell counts or HIV-1 viral loads. These observations reflect a difference in the distribution of HPV-16 variants among HIV-positive and -negative women, indicating that HIV-positive status may lead to increased prevalence of a subset of variants.

Detection of human papillomavirus DNA in urine specimens from human immunodeficiency virus-positive women.

Human immunodeficiency virus (HIV)-positive women may represent one of the fastest-growing populations at risk for acquiring cervical cancer and thus require frequent screening. The purpose of the present studies was to validate a PCR-based urine assay by comparing detection and genotyping of human papillomavirus (HPV) DNA in urine samples and matching cervical swab specimens of HIV-positive women. Despite a difference in amplifiability, the prevalence of any HPV genotype (58% for the cervical swab specimens and 48% for the urine specimens) was not significantly different in this population. The levels of concordance were 70, 71, and 78% for detection of any HPV type, any high-risk HPV type, or any low-risk HPV type in the two specimen types, respectively. While instances of discordant detection were greater for the cervical swab specimens than for the urine specimens, this was not statistically significant. The distributions of HPV genotypes were similar in the cervix and the urine for the majority of types examined. Importantly, detection of HPV DNA in urine was associated with an abnormal Papanicolaou smear to the same extent that detection of HPV DNA in a cervical swab specimen was. These data provide preliminary support for the proposal to use urine testing as a primary or secondary screening tool for cervical cancer in HIV-positive women or as an epidemiological tool. Additional studies with larger sample sizes must be conducted in order to further verify these findings.