Jocelyn M. Stewart

Phenotypic heterogeneity and instability of human ovarian tumor-initiating cells

The cancer stem cell (CSC) model proposes that tumors have a hierarchical organization in which only some cells indefinitely self-renew and thereby sustain tumor growth. In addition, the CSC model requires that tumor-initiating cells (TICs) be prospectively isolatable on the basis of their phenotype. Previous studies have suggested that serous ovarian cancer (SOC) conforms to the CSC model, but these used arguably nonfidelitous immortalized cell lines, cultured primary cells, or passaged xenografts as the source of tumor cells. We developed a robust assay for quantifying TICs from primary SOC. Using this assay, we find that TICs are rare when assayed in either NOD/SCID or NOD/SCID/IL2Rγ−/− (NSG) mice. TIC frequency (TICf) varies substantially between patients, although it is similar in primary ovarian masses and omental metastases, suggesting that TICf is an intrinsic property of ovarian tumors. CD133 marks all TICs from several primary SOC cases. However, in other cases, substantial TIC activity is found in both the CD133+ and CD133− fractions, whereas still other cases have exclusively CD133− TICs. Furthermore, the TIC phenotype can change in xenografts: primary tumors in which all TICs are CD133+ can give rise to xenografts that contain substantial numbers of CD133− TICs. Our results highlight the need for quantitative rigor in the evaluation of TICs and for caution when using passaged xenografts for such studies. Furthermore, although our data suggest that SOC conforms to the CSC hypothesis, the heterogeneity of the TIC phenotype may complicate its clinical application.

Comparison of annexin II, p63 and α‐methylacyl‐CoA racemase immunoreactivity in prostatic tissue: a tissue microarray study

Background: Current ancillary markers for diagnosis in prostate biopsies include p63 and α-methylacyl-CoA racemase (AMACR). Annexin II (ANXII), a calcium and phospholipid binding protein, is lost in prostate cancer. Aims: To investigate ANXII expression in order to assess its utility as a novel diagnostic marker in comparison to p63 and AMACR. Methods: Using immunohistochemistry on six tissue microarrays, ANXII, p63, and AMACR expression was analysed from 210 radical prostatectomy cases. Staining was evaluated in benign and atrophic glands, high-grade prostatic intraepithelial neoplasia (HGPIN), and prostatic adenocarcinoma. Separate scores were given for ANXII, AMACR and p63 expression. Results: Diffuse cytoplasmic expression of ANXII correlated with p63 reactivity in basal cells. Benign glands were positive for ANXII in 286/292 cores (98%) and negative for AMACR in all 292 cores. HGPIN showed heterogeneous expression of AMACR and ANXII. A significantly larger proportion of HGPIN glands were correctly identified as ANXII negative than as positive for AMACR. ANXII loss in prostate cancer was found in 282/320 cores (88%) and correlated with positive AMACR expression (272/320 cores, 85%), which was not statistically significant. There was no statistically significant correlation between ANXII scores and the clinical parameters examined. Conclusions: Immunohistochemical staining for ANXII is a consistent and reliable marker of prostatic neoplasia. The findings of this study suggest the potential utility of ANXII as a diagnostic aid in prostate cancer histopathology.

Biologically-Targeted Detection of Primary and Micro-Metastatic Ovarian Cancer

Ovarian cancer is the leading cause of morbidity/mortality from gynecologic malignancy. Early detection of disease is difficult due to the propensity for ovarian cancer to disseminate throughout the peritoneum. Currently, there is no single accurate test to detect primary or recurrent ovarian cancer. We report a novel clinical strategy using PPF: a multimodal, PET and optical, folate receptor (FR)-targeted agent for ovarian cancer imaging. The capabilities of PPF were evaluated in primary human ovarian cancer cells, in vivo xenografts derived from primary cells and ex vivo patient omemtum, as the heterogeneity and phenotype displayed by patients is retained. Primary cells uptake PPF in a FR-dependent manner demonstrating approximately a 5- to 25-fold increase in fluorescence. By both PET and fluorescence imaging, PPF specifically delineated FR-positive, ovarian cancer xenografts, with similar tumor-to-background ratios of 8.91±0.91 and 7.94±3.94, and micro-metastatic studding (<1mm), which demonstrated a 3.5-fold increase in PPF uptake over adjacent normal tissue. Ex vivo patient omentum demonstrated selective uptake of PFF by tumor deposits. The ability of PPF to identify metastatic deposits <1mm could facilitate more complete debulking (currently, optimal debulking is <10mm residual tumor), by providing a more sensitive imaging strategy improving treatment planning, response assessment and residual/recurrent disease detection. Therefore, PPF is a novel clinical imaging strategy that could substantially improve the prognosis of patients with ovarian cancer by allowing pre-, post- and intra-operative tumor monitoring, detection and possibly treatment throughout all stages of therapy and tumor progression.

Prognostic Significance of α-Methylacyl-CoA Racemase Among Men With High Grade Prostatic Intraepithelial Neoplasia in Prostate Biopsies

PURPOSE Serum prostate specific antigen screening has increased the number of prostate biopsies performed increasing the number of patients with high grade prostatic intraepithelial neoplasia. The criteria for re-biopsy are not standardized but may be refined by the identification of novel biomarkers demonstrating prognostic significance. Alpha-methylacyl-CoA racemase is a robust marker of prostate cancer and is expressed in a subset of high grade prostatic intraepithelial neoplasia. This study evaluates the prognostic significance of alpha-methylacyl-coA racemase positive high grade prostatic intraepithelial neoplasia glands in prostate biopsies. MATERIALS AND METHODS Immunohistochemical staining with alpha-methylacyl-coA racemase and p63 was examined in a selected group of 62 patients with a diagnosis of high grade prostatic intraepithelial neoplasia on initial prostate biopsy, of which on repeat biopsy 32 had no carcinoma and 30 had prostate cancer. There was no significant difference in age, number of cores sampled or prostate specific antigen history between the 2 outcome groups (ANOVA p >0.9). High grade prostatic intraepithelial neoplasia glands in each case were evaluated for alpha-methylacyl-coA racemase and p63. RESULTS Reactivity for alpha-methylacyl-coA racemase was found in 27 of the 62 cases examined. Fishers exact analysis revealed that patients with at least 1 alpha-methylacyl-coA racemase positive high grade prostatic intraepithelial neoplasia gland were 5.2 times more likely to have a subsequent diagnosis of prostate cancer on repeat biopsy than those without any alpha-methylacyl-coA racemase positive high grade prostatic intraepithelial neoplasia glands (p = 0.0044). No correlation was found between alpha-methylacyl-coA racemase positivity and any other clinical variable. CONCLUSIONS This is the first study to our knowledge to illustrate that alpha-methylacyl-coA racemase reactivity in high grade prostatic intraepithelial neoplasia may be useful to refine re-biopsy criteria and assist in clinical management decisions.

The optimal time for surgery in women with serous ovarian cancer.

BACKGROUND Advanced high-grade serous ovarian carcinoma (HGSC) is commonly treated with surgery and chemotherapy. We investigated the survival of patients treated with primary or interval surgery at different times following neoadjuvant chemotherapy. Their survival was compared with that of patients treated with primary cytoreductive surgery and adjuvant chemotherapy. METHODS Patients with stage III or IV HGSC were included in this retrospective cohort study. Clinical data were obtained from patient records. Patients were divided into 2 groups based on treatment with neoadjuvant chemotherapy and interval cytoreductive surgery (NAC) or with primary cytoreductive surgery and adjuvant chemotherapy (PCS). Study groups were stratified by several clinical variables. RESULTS We included 334 patients in our study: 156 in the NAC and 178 in the PCS groups. Survival of patients in the NAC group was independent of when they underwent interval cytoreductive surgery following initiation of neoadjuvant chemotherapy (p < 0.001). Optimal surgical cytoreduction had no impact on overall survival in the NAC group (p < 0.001). Optimal cytoreduction (p < 0.001) and platinum sensitivity (p < 0.001) were independent predictors of improved survival in the PCS but not in the NAC group. Patients in the NAC group had significantly worse overall survival than those in the PCS group (31.6 v. 61.3 mo, p < 0.001). CONCLUSION Women with advanced HGSC who underwent PCS had better survival than those who underwent interval NAC, regardless of the number of cycles of neoadjuvant therapy. Optimal cytoreduction did not provide a survival advantage in the NAC group.

The prognostic value of perioperative, pre‐systemic therapy CA125 levels in patients with high‐grade serous ovarian cancer

To investigate the ability of preoperative CA125 and post‐surgical CA125 changes to predict outcomes among patients with high‐grade serous ovarian cancer (HGSC).

A Genomically Characterized Collection of High-Grade Serous Ovarian Cancer Xenografts for Preclinical Testing

High-grade serous ovarian cancer (HGSC) is the leading cause of morbidity and mortality from gynecologic malignant tumors. Overall survival remains low because of the nearly ubiquitous emergence of platinum resistance and the paucity of effective next-line treatments. Current cell culture-based models show limited similarity to HGSC and are therefore unreliable predictive models for preclinical evaluation of investigational drugs. This deficiency could help explain the low overall rate of successful drug development and the decades of largely unchanged approaches to HGSC treatment. We used gene expression, copy number variation, and exome sequencing analyses to credential HGSC patient-derived xenografts (PDXs) as effective preclinical models that recapitulate the features of human HGSC. Mice bearing PDXs were also treated with standard-of-care carboplatin therapy. PDXs showed similar sensitivity to carboplatin as the patients tumor at the time of sampling. PDXs also recapitulated the diversity of genomic alterations (copy number variation and mutation profiles) previously described in large data sets that profiled HGSC. Furthermore, mRNA profiling showed that the PDXs represent all HGSC subtypes with the exception of the immunoreactive group. Credentialing of PDX models of HGSC should aid progress in HGSC research by providing improved preclinical models of HGSC that can be used to test novel targets and more accurately evaluate their likelihood of success.

Abstract B12: The role of stromal cells in supporting tumor-initiating cells in serous ovarian carcinoma

High grade serous ovarian carcinoma has been shown to be a highly heterogeneous disease. In vivo studies demonstrate that the tumor initiating frequency (TIF) varies substantially from case to case. Nonetheless, the tumor initiating subset remains a rare population within the epithelial compartment and can be enriched using the cell surface marker CD133 (Stewart et al., PNAS, 2011). The tumor microenvironment may play a role in supporting and maintaining tumor initiating cells (TIC) through direct and/ or indirect cross talk. The contribution of the mesenchymal component of the microenvironment may prove to be essential in providing such support as has been previously shown in studies on breast and prostate cancers. We hypothesize that cancer associated fibroblasts (CAFs) serve as a niche that supports and maintains the tumorigenic potential of TICs in SOC. We have derived and established CAF lines from bulk primary tumors and characterized their phenotype through immunostaining. Those lines stained positive for mesenchymal markers (Vimentin and Alpha-SMA), and stained negative for the epithelial marker Cytokertain. A profile of surface markers expressed on the surface of these CAF lines was then generated by running a flow cytometry-based high throughput screen (HTS) for 370 known cell surface proteins. Those markers are being validated for their specificity by immunostaining, FACS, qPCR, and in vivo work. Preliminary data obtained through immunoflourescnce and FACS support the specificity of our candidate stromal marker CD90. Furthermore, FACS data show that CD90 is expressed more brightly on fibroblasts than on epithelial cells (EpCAM+) in bulk SOC cases. Consequently, CD90 has been selected for sorting stromal cells for additional validation. Quantitative PCR analysis of CD90+EpCAM- sorted populations further validates their over-expression of mesenchymal genes when compared to CD90-EpCAM+ sorted populations. Moreover, functional validation of the influence of fibroblasts on the growth of tumor cells is currently being investigated through co-injection and co-culture assays. Preliminary data show that the presence of fibroblasts better supports the growth of epithelial colonies in culture than when compared to other conditions. Interplay between the niche and a specific subset of epithelial cells may promote those cells to become more tumorigenic. Such an interaction may be dependent on direct physical contact and/or indirect cross talk. Ultimately, we aim on understanding the mechanisms that govern these interactions. Citation Format: Ali Hussain, Jocelyn Stewart, Elzbieta Hyatt, Benjamin Neel, Laurie Ailles. The role of stromal cells in supporting tumor-initiating cells in serous ovarian carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B12.

Abstract SY35-02: Phenotypic heterogeneity and instability of human serous ovarian cancer tumor-initiating cells

Serous ovarian cancer (SOC) is the leading cause of morbidity/mortality from gynecologic malignancy. Current therapies increase survival significantly, yet the vast majority of SOC patients (70-90%) die of their disease. Patients almost always respond to initial courses of standard-of-care (platinum- and taxane-based) chemotherapy. Unfortunately, however, drug resistance almost always develops, resulting in the death of the patient. Two competing theories have been proposed to account for tumor initiation in SOC (and other malignancies); these theories also have important implications for therapy resistance. In the stochastic model, most, if not all, tumor cells can self-renew indefinitely; consequently, nearly all tumor cells provide a target population for the acquisition of drug resistance mutations. By contrast, the cancer stem cell (CSC) hypothesis holds that only a subset of tumor cells can initiate and maintain the tumor. Moreover, CSC might be intrinsically more resistant to some/many drugs that are effective against other (“bulk”) tumor cells. These two models differ fundamentally in their view of tumor-initiating cells (TIC). The essential feature of the CSC model is that tumors are organized hierarchically, such that TIC can be prospectively distinguished from non-TIC by phenotype. If the CSC model holds, it should be possible to identify and purify a stable cell population with the unique ability to generate serially transplantable tumors that recreate the heterogeneity of the initial malignancy. By contrast, the stochastic model predicts that TIC distribute into all cell fractions. Many studies of ovarian carcinogenesis and drug response have used immortalized cell lines grown for long periods of time in serum-containing culture. However, the extent to which these cells represent the biology of SOC is unclear. Many SOC lines do not reproduce serous histology when propagated as xenografts in immune-compromised mice; others cannot even give rise to xenografts. Even in culture, few of these lines show evidence of the cytologic and immunologic heterogeneity typically seen in primary tumors. Consequently, these lines might not be adequate for testing new therapies for SOC. Moreover, it is not clear that immortalized cell lines represent valid models for evaluating the CSC model or for studying TIC in SOC. Notably, studies of several other malignancies have shown that TIC as defined using immortalized cell lines do not have the same phenotype as those defined in xenografts using primary patient samples. Improving outcome for SOC patients will require better understanding of SOC pathogenesis and drug resistance, using assay systems that reflect the genetic and cellular diversity of human SOC more faithfully than conventional ovarian cancer cell lines. To this end, we have established a large collection of primary human SOC samples and developed a robust, quantitative assay for SOC tumor-initiating cells (TIC). Using this assay, we find that TICs are rare when assayed in either NOD/SCID or NOD/SCID/IL2Rγ −/− (NSG) mice. TIC frequency varies substantially between patients, although it is similar in primary ovarian masses and omental metastases, suggesting that TIC frequency is an intrinsic property of given ovarian tumor classes. For instance, CD133 marks all TICs from several primary SOC cases. However, in other cases, substantial TIC activity is found in both the CD133+ and CD133− fractions, whereas still other cases have exclusively CD133− TICs. Furthermore, the TIC phenotype can change in xenografts: primary tumors in which all TICs are CD133+ can give rise to xenografts that contain substantial numbers of CD133− TICs. Our results highlight the need for quantitative rigor in the evaluation of TICs and for caution when using passaged xenografts for such studies. Furthermore, although our data suggest that SOC conforms to the CSC hypothesis, the heterogeneity of the TIC phenotype may complicate its clinical application. To address whether instability in the TIC phenotype may be due to the acquisition of genetic alterations in the CD133- compared to the CD133+ fraction, we analyzed copy number alterations in CD133 positive and negative-derived xenografts. Preliminary analysis suggests that most genetic alterations are common in both fractions; however, in 3/6 cases, additional genetic alterations were observed in the CD133- fraction, suggesting the emergence of CD133- TIC might be, at least in part, genetically driven. The applicability of this observation to additional cases and primary sorted cells (i.e. those that were used to establish the xenografts), as well as the mechanism of genetic instability is currently under investigation. We used expression microarrays to examine differences between CD133+ and CD133- populations. We find that >1000 probes are differentially expressed in these two cell subsets, confirming that they are distinct cell populations. We have identified several putatively targetable pathways and transcription factor networks with altered expression in TIC-enriched fractions. The biological roles of these pathways in HG-SOC, specifically in the TIC compartment, are under investigation. Finally, we performed high-throughput flow cytometry: involving independent analysis of 365 cell surface markers to identify proteins expressed on all or subsets of HG-SOC cells. We have combined these analyses with mass cytometry (collaboration with Nolan9s lab, Stanford University). Mass cytometry allows deep profiling of cell attributes and function using a novel multi-parametric approach combining flow cytometry with mass spectrometry that allows examination of up to 35 cell surface and/or intracellular markers on a single cell. Examination of 40 primary ovarian cancer samples has provided further supportive evidence for inter- and intra-patient heterogeneity in HG-SOC. Current analyses focus on biological validation of identified subpopulations marked by a series of cell surface and putative “stem cell” gene sets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY35-02. doi:1538-7445.AM2012-SY35-02

Abstract 3318: Phenotypic heterogeneity and instability of tumor-initiating cells in high-grade serous cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Serous ovarian cancer (SOC) is the leading cause of morbidity/mortality from gynecologic malignancy. Current therapies significantly increase survival, yet 70-90% of SOC patients recur within five years and die of their disease. The cancer stem cell (CSC) hypothesis holds that only some tumor cells have the potential to initiate and maintain tumors. Moreover, CSC might be resistant to many therapeutic agents that are effective against bulk tumor cells, which could contribute to treatment failure. We have established a large collection of primary human SOC samples and developed a robust, quantitative assay for SOC tumor-initiating cells (TIC). Using this assay, we found that TIC from primary SOC can be identified based on their expression of the surface marker CD133. However, we also observed heterogeneity and instability of the TIC phenotype with disease progression. We examined whether instability in the TIC phenotype may be due to the acquisition of additional genetic alterations in the CD133- compared to the CD133+ fraction. Preliminary analysis suggests that most genetic alterations are common in both fractions; however, in 3/6 cases, additional genetic alterations were observed in the CD133- fraction, suggesting the emergence of CD133- TIC might be, at least in part, genetically driven. The mechanism of genetic instability is currently under investigation. In addition, we have used expression microarrays to examine differences between CD133+ and CD133- populations. We find that >1000 probes are differentially expressed in these two groups. We have identified several pathways with altered expression in TIC-enriched fractions. The biological role of these pathways in HG-SOC is under investigation. Our ability to identify and characterize TIC in this disease may facilitate development of new and more effective therapeutic strategies for ovarian cancer, thereby benefitting patients with this devastating disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3318. doi:1538-7445.AM2012-3318