Expanded evidence that the 31-gene expression profile test provides clinical utility for melanoma management in a multicenter study.

OBJECTIVE: National Comprehensive Cancer Network (NCCN) guidelines for cutaneous melanoma (CM) recommend physicians consider increased surveillance for patients who typically have lower melanoma survival rates (stages IIB-IV as determined by the American Joint Committee on Cancer (AJCC), 8th edition). However, up to 15% of patients identified as having a low recurrence risk (stages I-IIA) experience disease recurrence, and some patients identified as having a high recurrence risk will not experience any recurrence. The 31-gene expression profile test (31-GEP) stratifies patient recurrence risk into low (Class 1) and high (Class 2) and has demonstrated risk-appropriate impact on disease management and clinical decisions. METHODS: Five-year plans for lab work, frequency of clinical visits, and imaging pre- and post-31-GEP test results were assessed for a cohort of 509 stage I-III patients following an interim subset analysis of 247 patients. RESULTS: After receiving 31-GEP results, 50.6% of patients had a change in management plans in at least one of the following categories-clinical visits, lab work, or surveillance imaging. The changes aligned with the risk predicted by the 31-GEP for 76.1% of patients with a Class 1 result and 78.7% of patients with a Class 2 result. A Class 1 31-GEP result was associated with changes toward low-intensity management recommendations, while a Class 2 result was associated with changes toward high-intensity management recommendations. CONCLUSION: The 31-GEP can stratify patient recurrence risk in patients with CM, and clinicians understand and apply the prognostic ability of the 31-GEP test to alter patient management in risk-appropriate directions.

When caught early, cancer of the skin can usually be removed, and patients have excellent chances of survival. However, some patients will have their cancer come back or spread to a new location in their body.The 31-gene expression profile (GEP) test measures the expression levels of 31 genes from an individual patient's tumor. A proprietary formula uses this information to identify the risk of recurrence or spread as low risk (Class 1) or high risk (Class 2). Cancers with low-risk 31-GEP scores have a lower chance of cancer recurrence or spread than patients with a high-risk score.In this study, we wanted to determine if doctors treated patients with low-risk scores differently from patients with high-risk scores. We found that doctors changed approximately half of patient treatment plans (doctor visits, lab work, or imaging to see if the cancer has come back) after learning the 31-GEP test results. Doctors usually planned less frequent follow-up visits for Class 1 results and more frequent follow up for Class 2 results.This study found doctors understand and make changes to their treatment plans based on the patient's 31-GEP test result.

Development and validation of a nomogram incorporating gene expression profiling and clinical factors for accurate prediction of metastasis in patients with cutaneous melanoma following Mohs micrographic surgery.

BACKGROUND: There is a need to improve prognostic accuracy for patients with cutaneous melanoma. A 31-gene expression profile (31-GEP) test uses the molecular biology of primary tumors to identify individual patient metastatic risk. OBJECTIVE: Develop a nomogram incorporating 31-GEP with relevant clinical factors to improve prognostic accuracy. METHODS: In an IRB-approved study, 1124 patients from 9 Mohs micrographic surgery centers were prospectively enrolled, treated with Mohs micrographic surgery, and underwent 31-GEP testing. Data from 684 of those patients with at least 1-year follow-up or a metastatic event were included in nomogram development to predict metastatic risk. RESULTS: Logistic regression modeling of 31-GEP results and T stage provided the simplest nomogram with the lowest Bayesian information criteria score. Validation in an archival cohort (n = 901) demonstrated a significant linear correlation between observed and nomogram-predicted risk of metastasis. The resulting nomogram more accurately predicts the risk for cutaneous melanoma metastasis than T stage or 31-GEP alone. LIMITATIONS: The patient population is representative of Mohs micrographic surgery centers. Sentinel lymph node biopsy was not performed for most patients and could not be used in the nomogram. CONCLUSIONS: Integration of 31-GEP and T stage can gain clinically useful prognostic information from data obtained noninvasively.

Integrating 31-Gene Expression Profiling With Clinicopathologic Features to Optimize Cutaneous Melanoma Sentinel Lymph Node Metastasis Prediction.

National guidelines recommend sentinel lymph node biopsy (SLNB) be offered to patients with > 10% likelihood of sentinel lymph node (SLN) positivity. On the other hand, guidelines do not recommend SLNB for patients with T1a tumors without high-risk features who have < 5% likelihood of a positive SLN. However, the decision to perform SLNB is less certain for patients with higher-risk T1 melanomas in which a positive node is expected 5%-10% of the time. We hypothesized that integrating clinicopathologic features with the 31-gene expression profile (31-GEP) score using advanced artificial intelligence techniques would provide more precise SLN risk prediction. METHODS: An integrated 31-GEP (i31-GEP) neural network algorithm incorporating clinicopathologic features with the continuous 31-GEP score was developed using a previously reported patient cohort (n = 1,398) and validated using an independent cohort (n = 1,674). RESULTS: Compared with other covariates in the i31-GEP, the continuous 31-GEP score had the largest likelihood ratio (G2 = 91.3, P < .001) for predicting SLN positivity. The i31-GEP demonstrated high concordance between predicted and observed SLN positivity rates (linear regression slope = 0.999). The i31-GEP increased the percentage of patients with T1-T4 tumors predicted to have < 5% SLN-positive likelihood from 8.5% to 27.7% with a negative predictive value of 98%. Importantly, for patients with T1 tumors originally classified with a likelihood of SLN positivity of 5%-10%, the i31-GEP reclassified 63% of cases as having < 5% or > 10% likelihood of positive SLN, for a more precise, personalized, and clinically actionable SLN-positive likelihood estimate. CONCLUSION: These data suggest the i31-GEP could reduce the number of SLNBs performed by identifying patients with likelihood under the 5% threshold for performance of SLNB and improve the yield of positive SLNBs by identifying patients more likely to have a positive SLNB.

Selected microRNAs Increase Synaptic Resilience to the Damaging Binding of the Alzheimer's Disease Amyloid Beta Oligomers.

Alzheimer's disease (AD) is marked by synaptic loss (at early stages) and neuronal death (at late stages). Amyloid beta (Aß) and tau oligomers can target and disrupt synapses thus driving cognitive decay. Non-demented individuals with Alzheimer's neuropathology (NDAN) are capable of withstanding Aß and tau toxicity, thus remaining cognitively intact despite presence of AD neuropathology. Understanding the involved mechanism(s) would lead to development of novel effective therapeutic strategies aimed at promoting synaptic resilience to amyloid toxicity. NDAN have a unique hippocampal post-synaptic proteome when compared with AD and control individuals. Potential upstream modulators of such unique proteomic profile are miRNA-485, miRNA-4723 and miRNA-149, which we found differentially expressed in AD and NDAN vs. control. We thus hypothesized that these miRNAs play an important role in promoting either synaptic resistance or sensitization to Aß oligomer binding. Using an in vivo mouse model, we found that administration of these miRNAs affected key synaptic genes and significantly decreased Aß binding to the synapses. Our findings suggest that miRNA regulation and homeostasis are crucial for Aß interaction with synaptic terminals and support that a unique miRNA regulation could be driving synaptic resistance to Aß toxicity in NDAN, thus contributing to their preserved cognitive abilities.

Polymer-peptide delivery platforms: effect of oligopeptide orientation on polymer-based DNA delivery.

The success of nonviral transfection using polymers hinges on efficient nuclear uptake of nucleic acid cargo and overcoming intra- and extracellular barriers. By incorporating PKKKRKV heptapeptide pendent groups as nuclear localization signals (NLS) on a polymer backbone, we demonstrate protein expression levels higher than those obtained from JetPEI and Lipofectamine 2000, the latter being notorious for coupling high transfection efficiency with cytotoxicity. The orientation of the NLS peptide grafts markedly affected transfection performance. Polymers with the sequence attached to the backbone from the valine residue achieved a level of nuclear translocation higher than the levels of those having the NLS groups attached in the opposite orientation. The differences in nuclear localization and DNA complexation strength between the two orientations correlated with a striking difference in protein expression, both in cell culture and in vivo. Polyplexes formed from these comb polymer structures exhibited transfection efficiencies superior to those of Lipofectamine 2000 but with greatly reduced toxicity. Moreover, these novel polymers, when administered by intramuscular ultrasound-mediated delivery, allowed a high level of reporter gene expression in mice, demonstrating their therapeutic promise in vivo.

Human adipose-derived mesenchymal stromal cell pigment epithelium-derived factor cytotherapy modifies genetic and epigenetic profiles of prostate cancer cells.

BACKGROUND AIMS: Adipose-derived mesenchymal stromal cells (ASCs) are promising tools for delivery of cytotherapy against cancer. However, ASCs can exert profound effects on biological behavior of tumor cells. Our study aimed to examine the influence of ASCs on gene expression and epigenetic methylation profiles of prostate cancer cells as well as the impact of expressing a therapeutic gene on modifying the interaction between ASCs and prostate cancer cells. METHODS: ASCs were modified by lentiviral transduction to express either green fluorescent protein as a control or pigment epithelium-derived factor (PEDF) as a therapeutic molecule. PC3 prostate cancer cells were cultured in the presence of ASC culture-conditioned media (CCM), and effects on PC3 or DU145. Ras cells were examined by means of real-time quantitative polymerase chain reaction, EpiTect methyl prostate cancer-focused real-time quantitative polymerase chain reaction arrays, and luciferase reporter assays. RESULTS: ASCs transduced with lentiviral vectors were able to mediate expression of several tumor-inhibitory genes, some of which correlated with epigenetic methylation changes on cocultured PC3 prostate cancer cells. When PC3 cells were cultured with ASC-PEDF CCM, we observed a shift in the balance of gene expression toward tumor inhibition, which suggests that PEDF reduces the potential tumor-promoting activity of unmodified ASCs. CONCLUSIONS: These results suggest that ASC-PEDF CCM can promote reprogramming of tumor cells in a paracrine manner. An improved understanding of genetic and epigenetic events in prostate cancer growth in response to PEDF paracrine therapy would enable a more effective use of ASC-PEDF, with the goal of achieving safer yet more potent anti-tumor effects.

Interleukin-27 gene delivery for modifying malignant interactions between prostate tumor and bone.

We have examined the role of a novel cytokine, interleukin-27 (IL-27), in mediating interactions between prostate cancer and bone. IL-27 is the most recently characterized member of the family of heterodimeric IL-12-related cytokines and has shown promise in halting tumor growth and mediating tumor regression in several cancer models, including prostate cancer. Prostate cancer is frequently associated with metastases to the bone, where the tumor induces a vicious cycle of communication with osteoblasts and osteoclasts to induce bone lesions, which are a significant cause of pain and skeletal-related events for patients, including a high fracture risk. We describe our findings in the effects of IL-27 gene delivery on prostate cancer cells, osteoblasts, and osteoclasts at different stages of differentiation. We applied the IL-27 gene delivery protocol in vivo utilizing sonoporation (sonodelivery) with the goal of treating and reducing the growth of prostate cancer at a bone metastatic site in vivo. We used a new model of immune-competent prostate adenocarcinoma and characterized the tumor growth reduction, gene expression, and effector cellular profiles. Our results suggest that IL-27 can be effective in reducing tumor growth, can help normalize bone structure, and can promote enhanced accumulation of effector cells in prostate tumors. These results are promising, because they are relevant to developing a novel IL-27-based strategy that can treat both the tumor and the bone, by using this simple and effective sonodelivery method for treating prostate tumor bone metastases.

Interleukin-27 expression modifies prostate cancer cell crosstalk with bone and immune cells in vitro.

Prostate cancer is frequently associated with bone metastases, where the crosstalk between tumor cells and key cells of the bone microenvironment (osteoblasts, osteoclasts, immune cells) amplifies tumor growth. We have explored the potential of a novel cytokine, interleukin-27 (IL-27), for inhibiting this malignant crosstalk, and have examined the effect of autocrine IL-27 on prostate cancer cell gene expression, as well as the effect of paracrine IL-27 on gene expression in bone and T cells. In prostate tumor cells, IL-27 upregulated genes related to its signaling pathway while downregulating malignancy-related receptors and cytokine genes involved in gp130 signaling, as well as several protease genes. In both undifferentiated and differentiated osteoblasts, IL-27 modulated upregulation of genes related to its own signaling pathway as well as pro-osteogenic genes. In osteoclasts, IL-27 downregulated several genes typically involved in malignancy and also downregulated osteoclastogenesis-related genes. Furthermore, an osteogenesis-focused real-time PCR array revealed a more extensive profile of pro-osteogenic gene changes in both osteoblasts and osteoclasts. In T-lymphocyte cells, IL-27 upregulated several activation-related genes and also genes related to the IL-27 signaling pathway and downregulated several genes that could modulate osteoclastogenesis. Overall, our results suggest that IL-27 may be able to modify interactions between prostate tumor and bone microenvironment cells and thus could be used as a multifunctional therapeutic for restoring bone homeostasis while treating metastatic prostate tumors.

Pigment epithelial-derived factor and melanoma differentiation associated gene-7 cytokine gene therapies delivered by adipose-derived stromal/mesenchymal stem cells are effective in reducing prostate cancer cell growth.

Adipose-derived stromal/mesenchymal stem cells (ASC) have gained interest as promising tools for delivering cancer therapy. Adipose tissue can be obtained readily in amounts sufficient for ASC isolation, which can be expanded rapidly, allowing its use at low passage numbers, and can be transduced by viral and nonviral means. Our goal was to examine the potential of ASC to deliver cytokine gene therapies melanoma differentiation associated gene-7 (MDA-7) or pigment epithelial-derived factor (PEDF) to cancer cells. These novel cytokines are a potent proapoptotic and an antiangiogenesis mediator, respectively, with potential as antitumor agents. Expression of cytokine therapies did not adversely affect ASC biology, and these cells were still able to differentiate and retain normal viability. The ASC cytokine therapies were efficient in reducing tumor cell growth in coculture and also in suppressing in vitro angiogenesis phenotypes. We also observed that ASC retained their innate ability to migrate toward tumor cells in coculture, and this ability could be blocked by inhibition of CXCR4 signaling. The ASC were found to be nontumorigenic in vitro using a soft agar assay, as well as in vivo, utilizing 2 prostate cancer xenograft models. The ASC-MDA7 only reduced tumor growth in the TRAMP-C2-Ras (TC2Ras) prostate cancer model. The ASC-PEDF, however, reduced growth in both the TC2Ras and the PC3 highly aggressive prostate cancer models, and it was able to completely prevent prostate tumor establishment in vivo. In conclusion, ASC expressing PEDF and MDA7 could effectively reduce prostate tumor growth in vivo, suggesting ASC-cytokine therapies might have translational applications, especially the PEDF modality.

Advances in sonoporation strategies for cancer.

We will focus on the therapeutic applications of ultrasound (US) for gene transfection or 'sonoporation'. Sonoporation therapy or 'sonotherapy' is an emerging physical method for delivering drugs and/or nucleic acids for treating cancer. Because of its non-invasive nature, sonotherapy has the potential to be competitive with other treatment delivery methods such as viruses or lipofection. For nucleic acid delivery, sonoporation in the presence of microbubbles (MB) significantly enhances transfection efficiency. Sonoporation is an ideal means of delivering pDNA, and it has a similar efficiency as electroporation or other physical gene therapy techniques, with potentially fewer side effects. Typically, nonphysical means of gene delivery have suffered from lower efficiencies compared to viral vectors, however, several studies suggest that sonoporation pDNA delivery could be a simple and inexpensive method that only requires a plasmid, MB, and a sonoporation device. Sonoporation could also be used to target MB to certain cells/tissues, delivering localized therapies. Using high-performance probes, more precise and safer sonoporation treatments will be developed, and newer therapeutic prospects will be realized.

Sonoporation delivery of interleukin-27 gene therapy efficiently reduces prostate tumor cell growth in vivo.

We have examined the potential of a novel cytokine, interleukin-27 (IL-27), for gene therapy of prostate cancer. IL-27 is the most recently characterized member of the family of heterodimeric IL-12-related cytokines and has shown promise in halting tumor growth and mediating tumor regression in several cancer models. In the present study, we examined the efficacy of a new mode of gene delivery to prostate tumors: low-frequency ultrasound irradiation or "sonoporation." We also examined the potential of IL-27 gene delivery by sonoporation to treat and reduce the growth of prostate cancer in vivo. We used three models of immune-competent prostate adenocarcinoma and characterized the tumor-growth reduction, gene-profile expression, and effector cellular profiles. Our results suggest that IL-27 can be effective in reducing tumor growth and can help enhance accumulation of effector cells in prostate tumors in vivo. These results are promising, because they are potentially relevant to developing novel therapies that can be translated by using the novel and effective sonoporation gene-therapy delivery strategy.

Cell-cycle regulators cdk2ap1 and bicalutamide suppress malignant biological interactions between prostate cancer and bone cells.

INTRODUCTION: We examined whether the novel cell-cycle regulator cdk2-associated protein 1 (p12(cdk2ap1) or cdk2ap1), recently shown to regulate prostate cancer cell cycle and apoptosis, could have the capacity to reduce invasiveness and/or reduce malignant biological interactions between prostate cancer and bone cells. We also examined whether combining two cell-cycle arrest stimuli, cdk2ap1 plus bicalutamide (or casodex, CDX), could help enhance inhibition of prostate cancer cell phenotypes. METHODS: We stably expressed cdk2ap1 in prostate cancer cell lines using lentiviral vectors, as well as several different co-culture assays to quantify cellular invasion, migration, and the effect of the treatments on interaction with the bone microenvironment. RESULTS: We have determined that cdk2ap1 can further augment the effects of CDX on cell-cycle arrest, growth inhibition, and cellular invasion. Using a coculture model, we observed that either cdk2ap1 or cdk2ap1/CDX combination were able to reduce chemotaxis towards osteoblasts, and also reduce the osteoblastic proliferative response to prostate cancer. Also modified by cdk2ap1 and CDX were several signaling pathways associated with prostate cancer/bone crosstalk mechanisms involved in prostate cancer progression. CONCLUSIONS: These results suggest that either cdk2ap1 or the cdk2ap1/CDX combination hold promise in regulating prostate cancer growth and malignant phenotypes, and potentially also in reducing procarcinogenic interactions with a bone microenvironment model, restoring malignant phenotypes and signaling to a more benign state.

Novel tumor growth inhibition mechanism by cell cycle regulator cdk2ap1 involves antiangiogenesis modulation.

We evaluated the effect of expressing the cell cycle regulator protein cdk2-associating protein1 (cdk2ap1) in inhibiting growth of squamous cell carcinoma (SCC). Expression of cdk2ap1 correlated with reduction in several SCC malignant cell phenotypes, including reduced angiogenesis. We observed several alterations in gene expression consistent with classical functions of cdk2ap1, including upregulation of cell cycle inhibitory genes, and an upregulation in expression of genes belonging to both intrinsic and extrinsic apoptotic cascades. Interestingly, we also uncovered a profile of gene expression and activation of signaling pathways that may suggest new tumor-suppressive functions for cdk2ap1 through downregulation of invasion/metastasis and modulation of antiangiogenesis by upregulation of the TGFß signaling pathway. Blocking of the TGFß1 pathway resulted in inhibition of the cdk2ap1 antiangiogenesis phenotype. In combination, these data support the role of cdk2ap1 as a tumor suppressor gene that can regulate SCC tumor growth in a cell autonomous manner through decreases in invasiveness and a non-cell autonomous manner through decreases in angiogenesis phenotypes, and these are novel phenotypes induced by cdk2ap1.

Cell cycle regulator cdk2ap1 inhibits prostate cancer cell growth and modifies androgen-responsive pathway function.

BACKGROUND: We evaluated the effect of expressing the cell cycle regulator cdk2ap1, downregulated in prostate cancer cell lines, in inhibiting prostate cancer cell growth. METHODS: Expression of cdk2ap1 using a tet-inducible lentiviral system modified growth rate, induced cell cycle arrest and apoptosis and reduced the invasive ability of prostate cancer cell lines, as assayed by cell viability, cell cycle profiling, Caspase 3/7 detection, and matrigel invasion assays. We examined the effect of expressing cdk2ap1 on gene expression profiles of cytokine, invasion, apoptotic, and androgen response pathways using quantitative real-time PCR, and used androgen-responsive reporter gene assays, and methylation-sensitive PCR to examine the mechanism of cdk2ap1 interaction with androgen-responsive pathways. RESULTS: The expression of cdk2ap1 correlated with a reduction in cellular growth, irrespective of inhibition or stimulation of androgen receptor (AR) signaling pathways. Cell cycle arrest, increased apoptosis, and a reduction in invasiveness phenotypes were observed upon cdk2ap1 expression. Enhanced demethylation at the AR promoter, AR expression increases, and enhanced AR transcriptional activity correlated with cdk2ap1 expression. CONCLUSIONS: Our findings support a novel concept by which cell cycle inhibitor genes can impact prostate cancer phenotypes by restoring a tumor suppressive function to androgen-responsive pathways and this function may involve modulation of a subset of functions of the AR.

Expression of cell cycle regulator cdk2ap1 suppresses tumor cell phenotype by non-cell-autonomous mechanisms.

We evaluated the effect of expressing the cell cycle regulator cdk2ap1 in epithelial or stromal cell compartments to reduce SCC growth in vitro and in vivo. Cell-autonomous and/or non-cell-autonomous expression of cdk2ap1 reduced tumor growth and invasion and altered cell cycle, adhesion, invasion, angiogenesis, and apoptotic gene expression, as assessed by several in vitro phenotype assays, quantitative real-time PCR, and in vivo molecular imaging using a novel three-way xenograft animal model. Our findings suggest that the interactions between cancer cells and fibroblasts that promote abnormal growth can be minimized by expressing cdk2ap1, supporting a novel concept by which tumor/growth suppressor genes can impact tumorigenesis phenotypes from non-cell-autonomous interactions within the tumor microenvironment.