Examination of the engraftment of exogenous mesenchymal stem cells in ovarian tumors and their potential use as delivery vehicles for therapeutic genes

Interactions between neoplastic cells and the host stroma play a role in both tumor cell migration and proliferation. Stromal cells provide structural support for malignant cells, modulate the tumor microenvironment, and influence phenotypic behavior as well as the aggressiveness of the malignancy. In response, the tumor provides growth factors, cytokines, and cellular signals that continually initiate new stromal reactions and recruit new cells into the microenvironment to further support tumor growth. Since growing tumors recruit local cells, as well as supplemental cells from the circulation, such as fibroblasts and endothelial precursors, the question arises if it would be possible to access circulating stromal cells to modify the tumor microenvironment for therapeutic benefits. One such cell type, mesenchymal stem cells (MSC), could theoretically be engrafted into stroma. MSC are pluripotent cells that have been shown to form stromal elements such as myofibroblasts, perivascular tissues and connective tissues. Several reports have demonstrated that MSC can incorporate into sites of wound healing and tissue repair, due to active tissue remodeling and local paracrine factors, and given the similarity between wound healing and the carcinoma induced stromal response one can hypothesize that MSC have the potential to be recruited to sites of tumor development. In addition, gene-modified MSC could be used as cellular vehicles to deliver gene products into tumors. My results indicate that MSC home to and participate in tumor stroma formation in ovarian tumor xenografts in mice. Additionally, once homed to tumor beds, MSC proliferate rapidly and integrate. My studies aim at understanding the fate of MSC in the tumor microenvironment, as well as utilizing them for cellular delivery of therapeutic genes into the stroma of ovarian carcinomas. ^

Response to chemotherapy, recurrence and survival in advanced-stage ovarian, fallopian tube and primary peritoneal cancer patients with non-Ashkenazi Jewish BRCA mutations, compared to those without

Objectives. Previous studies have shown a survival advantage in ovarian cancer patients with Ashkenazi-Jewish (AJ) BRCA founder mutations, compared to sporadic ovarian cancer patients. The purpose of this study was to determine if this association exists in ovarian cancer patients with non-Ashkenazi Jewish BRCA mutations. In addition, we sought to account for possible "survival bias" by minimizing any lead time that may exist between diagnosis and genetic testing. ^ Methods. Patients with stage III/IV ovarian, fallopian tube, or primary peritoneal cancer and a non-Ashkenazi Jewish BRCA1 or 2 mutation, seen for genetic testing January 1996-July 2007, were identified from genetics and institutional databases. Medical records were reviewed for clinical factors, including response to initial chemotherapy. Patients with sporadic (non-hereditary) ovarian, fallopian tube, or primary peritoneal cancer, without family history of breast or ovarian cancer, were compared to similar cases, matched by age, stage, year of diagnosis, and vital status at time interval to BRCA testing. When possible, 2 sporadic patients were matched to each BRCA patient. An additional group of unmatched, sporadic ovarian, fallopian tube and primary peritoneal cancer patients was included for a separate analysis. Progression-free (PFS) & overall survival (OS) were calculated by the Kaplan-Meier method. Multivariate Cox proportional hazards models were calculated for variables of interest. Matched pairs were treated as clusters. Stratified log rank test was used to calculate survival data for matched pairs using paired event times. Fisher's exact test, chi-square, and univariate logistic regression were also used for analysis. ^ Results. Forty five advanced-stage ovarian, fallopian tube and primary peritoneal cancer patients with non-Ashkenazi Jewish (non-AJ) BRCA mutations, 86 sporadic-matched and 414 sporadic-unmatched patients were analyzed. Compared to the sporadic-matched and sporadic-unmatched ovarian cancer patients, non-AJ BRCA mutation carriers had longer PFS (17.9 & 13.8 mos. vs. 32.0 mos., HR 1.76 [95% CI 1.13–2.75] & 2.61 [95% CI 1.70–4.00]). In relation to the sporadic- unmatched patients, non-AJ BRCA patients had greater odds of complete response to initial chemotherapy (OR 2.25 [95% CI 1.17–5.41]) and improved OS (37.6 mos. vs. 101.4 mos., HR 2.64 [95% CI 1.49–4.67]). ^ Conclusions. This study demonstrates a significant survival advantage in advanced-stage ovarian cancer patients with non-AJ BRCA mutations, confirming the previous studies in the Jewish population. Our efforts to account for "survival bias," by matching, will continue with collaborative studies. ^

Role of ovarian cancer antigen CA125/MUC16 in development and cancer

Cancer antigen 125 (CA125) is a tumor antigen that is routinely used to monitor the disease progress and the outcome of treatment in ovarian cancer patients. Elevated serum levels of CA125 are detected in over 80% of epithelial ovarian cancer patients. CA125 is a high molecular weight (>1M Dalton) mucin-type glycoprotein encoded by the MUC16 gene on human chromosome 19. Although MUC16 has served as the best serum marker for monitoring growth of ovarian cancer, roles for MUC16 in normal physiology and ovarian cancer are largely unknown. To understand the biological functions of MUC16, I characterized a mouse Muc16 homolog on chromosome 9 by means of expression pattern profiling, phenotype analysis of Muc16 knockout mice, and in vitro and in vivo studies of Muc16 null transformed ovarian surface epithelial (OSE) cells. ^ The mouse Muc16 homolog shares a conserved genomic structure with human MUC16. In addition to being expressed in mouse ovarian cancer, mouse Muc16 mRNA and protein were expressed in the mesothelia covering the heart, lung, ovary, oviduct, spleen, testis, and uterus. The conserved genomic structure and expression pattern of mouse Muc16 to human MUC16 suggests that mouse Muc16 is the ortholog of human MUC16. To understand the biological functions of Muc16, I generated Muc16 knockout mice. Muc16 knockout mice were viable, fertile and normal by one year of age. However, between 18 and 24 months of age, Muc16 knockout mice developed various tissue abnormalities such as ovarian cysts and tumors of the liver and other peritoneal organs. To determine the role of MUC16 in ovarian cancer progression, I established Muc16 null transformed ovarian surface epithelial (OSE) cell lines, following the same method to develop mouse model of epithelial ovarian cancer (Orsulic et al., 2002). Loss of Muc16 did not affect cell morphology, cell proliferation rate, or tumorigenic potential. However, Muc16-null OSE cells showed decreased attachment to extracellular matrix proteins as well as to primary mouse peritoneal mesothelial cells. Peritoneal mesothelia are the most frequent implantation sites of ovarian cancer. Furthermore, a pilot transplantation assay suggests that Muc16 null transformed OSE cells formed less disseminated tumors in the peritoneal cavity compared to wild-type OSE cells. ^ In conclusion, these results demonstrate that MUC16 is not required for normal mouse development or reproduction, but plays important roles in tissue homeostasis, ovarian cancer cell adhesion and dissemination. This study provides the first in vivo evidence of the roles of MUC16 in development, as well as ovarian cancer progression and dissemination. These studies offer valuable insights into possible mechanisms of ovarian cancer development and potential molecular targets for ovarian cancer treatment. ^

The role of cardiovascular comorbidities in ovarian cancer survival

Objective. One facet of cancer care that often goes ignored is comorbidities, or diseases that exist in concert with cancer. Comorbid conditions may affect survival by influencing treatment decisions and prognosis. The purpose of this secondary data analysis was to identify whether a history of cardiovascular comorbidities among ovarian cancer patients influenced survival time at the University of Texas M. D. Anderson Cancer Center. The parent study, Project Peace, has a longitudinal design with an embedded randomized efficacy study which seeks to improve detection of depressive disorders in ovarian, peritoneal, and fallopian tube cancers. ^ Methods. Survival time was calculated for the 249 ovarian cancer patients abstracted by Project Peace staff. Cardiovascular comorbidities were documented as present, based upon information from medical records in addition to self reported comorbidities in a baseline study questionnaire. Kaplan-Meier survival curves were used to compare survival time among patients with a presence or absence of particular cardiovascular comorbidities. Cox Regression proportional models accounted for multivariable factors such as age, staging, family history of cardiovascular comorbidities, and treatment. ^ Results. Among our patient population, there was a statistically significant relationship between shorter survival time and a history of thrombosis, pericardial disease/tamponade, or COPD/pulmonary hypertension. Ovarian cancer patients with a history of thrombosis lived approximately half as long as patients without thrombosis (58.06 months vs. 121.55 months; p=.001). In addition, patients who suffered from pericardial disease/tamponade had poorer survival than those without a history of pericardial disease/tamponade (48 months vs. 80.07 months; p=.002). Ovarian cancer patients with a history of COPD or pulmonary hypertension had a median survival of 60.2 months, while the median survival for patients without these comorbidities was 80.2 months (p=.014). ^ Conclusion. Especially because of its relatively lower survival rate, greater emphasis needs to be placed on the potential influence of cardiovascular comorbid conditions in ovarian cancer.^

Quality of life, symptom experience, and use of complementary and alternative medicine among women with ovarian cancer

The objectives of this dissertation were to determine the quality of life in women with ovarian cancer and the association of their physical and emotional well-being with the number of symptoms, duration of symptoms, and the scores of common symptoms of ovarian cancer; to study the prevalence of complementary and alternative medicine techniques for symptom relief and its association with the number of symptoms, age, education, insurance, comorbidity, and satisfaction with medical care they received, and their pre-diagnostic experience of symptoms.^ This study was based on a secondary data analysis of a study of early detection of ovarian cancer. A sample of 139 women with ovarian cancer was recruited and was administered a questionnaire comprised of questions on their quality of life, their symptoms and what they did about the symptoms, whether they used any complementary and alternative medicine techniques, and other medical conditions they had. Out of this sample, 53 patients underwent in-depth interviews relating to their symptoms before the diagnosis and their experiences with the health care system leading to the ovarian cancer diagnosis. ^ In article #1, ovarian cancer patients were observed to have significantly poorer quality of life on all subscales and summary scores except pain, compared to that of the general population of US women. Physical well-being scores were negatively associated with the number of symptoms before diagnosis and a significant negative association of comorbidity index was observed with physical well-being. Higher education and increase in time since diagnosis was found to have better physical scores. Emotional well-being scores showed marginally significant associations with number of symptoms and bloating. ^ In article #2, a thematic content analysis of the ovarian cancer patients’ interviews revealed that on recognition of their symptoms women first assumed their symptoms to be a normal transient occurrence due to a pre-existing disease condition, or due to some other disease. A series of misattributions of their symptoms on their and their doctors’ part impacted their health care seeking.In article #3, a significantly greater likelihood of CAM use with an increase in the number of symptoms was observed.^ Based on the foregoing results, it is important to educate women on possible signs of ovarian cancer and also to educate doctors about the results of current research regarding ovarian cancer diagnosis. This will help to avoid a delay in getting a diagnosis and improve women’s quality of life. It emphasizes the diagnosis of ovarian cancer in earlier stages by more sensitive screening techniques. This study emphasizes the importance of consideration of comorbidity in any quality of life research. Additionally, educating women in the safe use of CAM techniques carries immense significance because the efficacy and safety of many of the currently advertized CAM products has not been scientifically validated. Further research is needed to confirm the findings of this study. ^

Genetic variants in the mammalian target of rapamycin (mTOR) signaling pathway as predictors of survival and clinical response in women with ovarian cancer

Background. The mTOR pathway is commonly altered in human tumors and promotes cell survival and proliferation. Preliminary evidence suggests this pathway's involvement in chemoresistance to platinum and taxanes, first line therapy for epithelial ovarian cancer. A pathway-based approach was used to identify individual germline single nucleotide polymorphisms (SNPs) and cumulative effects of multiple genetic variants in mTOR pathway genes and their association with clinical outcome in women with ovarian cancer. ^ Methods. The case-series was restricted to 319 non-Hispanic white women with high grade ovarian cancer treated with surgery and platinum-based chemotherapy. 135 SNPs in 20 representative genes in the mTOR pathway were genotyped. Hazard ratios (HRs) for death and Odds ratios (ORs) for failure to respond to primary therapy were estimated for each SNP using the multivariate Cox proportional hazards model and multivariate logistic regression model, respectively, while adjusting for age, stage, histology and treatment sequence. A survival tree analysis of SNPs with a statistically significant association (p<0.05) was performed to identify higher order gene-gene interactions and their association with overall survival. ^ Results. There was no statistically significant difference in survival by tumor histology or treatment regimen. The median survival for the cohort was 48.3 months. Seven SNPs were significantly associated with decreased survival. Compared to those with no unfavorable genotypes, the HR for death increased significantly with the increasing number of unfavorable genotypes and women in the highest risk category had HR of 4.06 (95% CI 2.29–7.21). The survival tree analysis also identified patients with different survival patterns based on their genetic profiles. 13 SNPs on five different genes were found to be significantly associated with a treatment response, defined as no evidence of disease after completion of primary therapy. Rare homozygous genotype of SNP rs6973428 showed a 5.5-fold increased risk compared to the wild type carrying genotypes. In the cumulative effect analysis, the highest risk group (individuals with ≥8 unfavorable genotypes) was significantly less likely to respond to chemotherapy (OR=8.40, 95% CI 3.10–22.75) compared to the low risk group (≤4 unfavorable genotypes). ^ Conclusions. A pathway-based approach can demonstrate cumulative effects of multiple genetic variants on clinical response to chemotherapy and survival. Therapy targeting the mTOR pathway may modify outcome in select patients.^

Identification of micro-RNAs targeting genes of PI3K/AKT pathway in ovarian cancer

Ovarian cancer is the leading cause of cancer-related death for females due to lack of specific early detection method. It is of great interest to find molecular-based biomarkers which are sensitive and specific to ovarian cancer for early diagnosis, prognosis and therapeutics. miRNAs have been proposed to be potential biomarkers that could be used in cancer prevention and therapeutics. The current study analyzed the miRNA and mRNA expression data extracted from the Cancer Genome Atlas (TCGA) database. Using simple linear regression and multiple regression models, we found 71 miRNA-mRNA pairs which were negatively associated between 56 miRNAs and 24 genes of PI3K/AKT pathway. Among these miRNA and mRNA target pairs, 9 of them were in agreement with the predictions from the most commonly used target prediction programs including miRGen, miRDB, miRTarbase and miR2Disease. These shared miRNA-mRNA pairs were considered to be the most potential genes that were involved in ovarian cancer. Furthermore, 4 of the 9 target genes encode cell cycle or apoptosis related proteins including Cyclin D1, p21, FOXO1 and Bcl2, suggesting that their regulator miRNAs including miR-16, miR-96 and miR-21 most likely played important roles in promoting tumor growth through dysregulated cell cycle or apoptosis. miR-96 was also found to directly target IRS-1. In addition, the results showed that miR-17 and miR-9 may be involved in ovarian cancer through targeting JAK1. This study might provide evidence for using miRNA or miRNA profile as biomarker.^

Platelets and Anti-Angiogenic Resistance in Ovarian Carcinoma

Background: Resistance to targeted anti-angiogenic therapy is a growing clinical concern given the disappointing clinical impact of anti-angiogenic. Platelets represent a component of the tumor microenvironment that are implicated in metastasis and represent a significant reservoir of angiogenic regulators. Thrombocytosis has been shown to be caused by malignancy and associated with adverse clinical outcomes, however the causal connections between these associations remain to be identified. Materials and Methods: Following IRB approval, patient data were collected on patients from four U.S. centers and platelet levels through and after therapy were considered as indicators of recurrence of disease. In vitro effects of platelets on cancer cell proliferation, apoptosis, and migration were examined. RNA interference was used to query signaling pathways mediating these effects. The necessity of platelet activation for in vitro effect was analyzed. In vivo orthotopic models were used to query the impact of thrombocytosis and thrombocytopenia on the efficacy of cytotoxic chemotherapy, the effect of aspirin on thrombocytosis and cancer, and platelet effect on anti-angiogenic therapy. Results: Platelets were found to increase at the time of diagnosis of ovarian cancer recurrence in a pattern comparable to CA-125. Platelet co-culture increased proliferation, increased migration, and decreased apoptosis in all cell lines tested. RNA interference implicated platelet derived growth factor alpha (PDGFRA) and transforming growth factor beta-receptor 1 (TGFBR1) signaling. Biodistribution studies suggested minimal platelet sequestration of taxanes. Blockade of platelet activation blocked in vitro effects. In vivo, thrombocytosis blocked chemotherapeutic efficacy, thrombocytopenia increased chemotherapeutic efficacy, and aspirin therapy partially blocked the effects of thrombocytosis. In vivo, withdrawal of anti-angiogenic therapy caused loss of therapeutic benefit with evidence of accelerated disease growth. This effect was blocked by use of a small-molecule inhibitor of Focal Adhesion Kinase. Anti-angiogenic therapy was also associated with increased platelet infiltration into tumor that was not seen to the same degree in the control or FAK-inhibitor-treated mice. Conclusions: Platelets are active participants in the growth and metastasis of tumor, both directly and via facilitation of angiogenesis. Blocking platelets, blocking platelet activation, and blocking platelet trafficking into tumor are novel therapeutic avenues supported by this data. Copyright © 2012 Justin Neal Bottsford-Miller, all rights reserved.

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE Publication No.________Kiera Rycaj, B.S.Supervisory Professor: Feng Wang-Johanning, Ph.D., M.D. Ovarian cancer (OC) is the fourth most common cancer in women, and the most lethal gynecologic malignancy in the United States. Adequate screening methodologies are currently lacking and most women first present with either stage III or IV disease. To date, there has been no substantial decrease in death rates and the majorities of patients relapse and die from their disease despite response to first-line therapy. Several proteins, such as CA-125, are elevated in OC, but none has proven specific and sensitive enough to serve as a screening tool or for tumor cell recognition and lysis. It has been proposed that human endogenous retrovirus sequences (HERVs) may play a role in the etiology of certain cancers. In a previous study, we showed that HERV-K envelope (env) proteins are widely expressed in human invasive breast cancer (BC) and ductal carcinoma in situ (DCIS), and elicit both serologic and cell-mediated immune responses in BC patients. We also reported the expression of multiple HERV genes and proteins in OC cell lines and tissues. In this study, we strengthened our previous data by determining that HERV-K env mRNAs are expressed in 69% of primary OC tissues (n=29), but in only 24% of benign tissues (N=17). Immmunohistochemistry (IHC) staining revealed HERV-Kpositivecancer cells detected in endometrioid adenocarcinoma and serous adenocarcinoma but not in benign cyst or normal epithelium biopsies. Immunofluorescence staining (IFS) showed greater cell surface expression of HERV-K in OC samples compared to adjacent uninvolved samples. Enzyme-linked immunosorbent assay (ELISA) data confirmed that a humoral immune response is elicited against HERV-K in OC patients. T-cell responses against HERV-K in lymphocytes from OC patients stimulated with autologous HERV-K pulsed dendritic cells included induction of T-cell proliferation and IFN-γ production. HERV-K–specific cytolytic T cells induced greater specific lysis of OC target cells compared to benign and adjacent uninvolved target cells. Finally, upon T regulatory cell (T-reg) depletion, 64% of OC patients displayed an increase in the specific lysis of target cells expressing HERV-K env protein. These findings suggest that HERV-K env protein is a tumor-associated antigen capable of activating both T-cell and B-cell responses in OC patients, and has great potential in the development of immunotherapy regimens against OC.

A systematic review and analysis of the association between endometriosis and ovarian cancer

Ovarian cancer is the most lethal of the gynecologic malignancies. The development of endometriosis has been shown to increase one's risk of ovarian cancer. Numerous studies have investigated this association, yet none have synthesized the available data. In a pooled analysis of cohort and case-control studies, the association between endometriosis and ovarian cancer was strengthened. Women who developed endometriosis-associated ovarian cancer were more likely to develop an early stage clear cell or endometrioid ovarian cancer histotypes and were more likely to have a better overall prognosis. The prognostic differences between endometriosis-associated ovarian cancer and ovarian cancer without an associated endometriosis may indicate genetic and environmental differences between groups.^

The effects of IL -10 producing monocytes on T-cell activation in patients with epithelial ovarian cancer

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites of patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-β2, but not IL-12, IL-1α, or TNF-α, and expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a, CD11b, or CD25 cell surface antigens. Since this subset of monocytes could affect the modulation of tumor immune responses in vivo, studies were undertaken to determine their effect on the activation and proliferation of autologous T-cells from the peritoneal cavity of patients with ovarian carcinoma. Cytokine transcripts, including IL-2, GM-CSF, and IFN-γ were detected in T-cells isolated from patient specimens that also contained the IL-10 producing monocytes, although the IFN-γ and IL-2 proteins could not be detected in T-cells co-incubated with the IL-10 producing monocytes in vitro. Additionally, IL-10 producing monocytes co-cultured with autologous T-cells inhibited the proliferation of the T-cells in response to PHA. T-cell proliferation and cytokine protein production could be restored by the addition of neutralizing antibodies to IL-10R and TGF-β to the co-culture system. These results suggested that this subset of monocytes may modulate antitumor immune responses by inhibiting T-cell proliferation and cytokine protein production. Further studies determined that the precursors to the inhibitory monocytes were tumor-associated and only present in the peripheral blood of patients with ovarian cancer and not present in the peripheral blood of healthy donors. These precursors could be induced to the suppressor phenotype by the addition of IL-2 and GM-CSF, two cytokines detected in the peritoneal cavity of ovarian cancer patients. Lastly, it was shown that the suppressor monocytes from the peritoneal cavity of ovarian cancer patients could be differentiated to a non-inhibitory phenotype by the addition of TNF-α and IFN-γ to the culture system. The differentiated monocytes did not produce IL-10, expressed the activation antigens HLA-DR, CD80, and CD86, and were able to stimulate autologous T-cells in vitro. Since a concomitant reduction in immune function is associated with tumor growth and progression, the effects of these monocytes are of considerable importance in the context of tumor immunotherapy. ^

Defective antitumor function of monocyte -derived macrophages from epithelial ovarian cancer patients

An abundance of monocytes and macrophages (MO/MA) in the microenvironment of epithelial ovarian cancer (EOC) suggests possible dual roles for these cells. Certain MO/MA subpopulations may inhibit tumor growth by antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or stimulation of adaptive immunity. In contrast, other MO/MA subpopulations may support tumor growth by immunosuppressive or pro-angiogenic cytokine production. A better understanding of the phenotype and activity of MO/MA in EOC should lead to greater insight into their role in the immunopathobiology of EOC and hence suggest targets for treatment. We have found differences in the proportions of MO/MA subpopulations in the peripheral blood and ascites of EOC patients compared to normal donors, and differences in MO/MA surface phenotype in the associated tumor environment compared to the systemic circulation. We also demonstrate that, following their activation in vitro, monocyte-derived macrophages (MDM) from the peripheral blood and ascites of EOC patients exhibit antitumor effector activities that are different from the behavior of normal donor cells. The phenotypic characteristics and antitumor activity of CD14+ MO/MA and an isolated subpopulation of CD14brightCD16 −HLA-DR+ MO/MA were compared in samples of normal donor peripheral blood and the peripheral blood and ascites from EOC patients. MDM were cultured with macrophage colony-stimulating factor (M-CSF) and activated with lipopolysaccharide (LPS) or a combination of LPS plus recombinant interferon-gamma. We determined that MO/MA from EOC patients had altered morphology and significantly less ADCC and phagocytic activity than did MO/MA from normal donors. ADCC and phagocytosis are mediated by receptors for the Fe portion of IgG (FcγRs), the expression of which were also found to be deficient on EOC MDM from peripheral blood and ascites. Anti-tumor functions not mediated by the FcγRs, such as macrophage mediated cytotoxicity and cytostasis, were not impaired in EOC MDM compared to normal donor MDM. Our findings also showed that MDM from both EOC patients and normal donors produce M-CSF-stimulated cytokines, including interleukin-8, tumor necrosis factor alpha, and interleukin-6, which have the potential to support ovarian tumor growth and metastasis. These findings may be relevant to the pathogenesis of EOC and to the development of future bioimmunotherapeutic strategies. ^