Strategies For Future Clinical Trials Research Articles

Noninvasive brain stimulation in autism: review and outlook for personalized interventions in adult patients.

Noninvasive brain stimulation (NIBS) has been increasingly investigated during the last decade as a treatment option for persons with autism spectrum disorder (ASD). Yet, previous studies did not reach a consensus on a superior treatment protocol or stimulation target. Persons with ASD often suffer from social isolation and high rates of unemployment, arising from difficulties in social interaction. ASD involves multiple neural systems involved in perception, language, and cognition, and the underlying brain networks of these functional domains have been well documented. Aiming to provide an overview of NIBS effects when targeting these neural systems in late adolescent and adult ASD, we conducted a systematic search of the literature starting at 631 non-duplicate publications, leading to six studies corresponding with inclusion and exclusion criteria. We discuss these studies regarding their treatment rationale and the accordingly chosen methodological setup. The results of these studies vary, while methodological advances may allow to explain some of the variability. Based on these insights, we discuss strategies for future clinical trials to personalize the selection of brain stimulation targets taking into account intersubject variability of brain anatomy as well as function.

Abstract A003: A novel treatment strategy for high-risk and relapse/refractory hepatoblastoma

Abstract Background: Patients with metastatic, treatment-refractory, and relapsed hepatoblastoma (HB) have survival rates of less than 50% due to limited treatment options. To better understand these aggressive phenotypes, our lab has developed a preclinical testing pipeline consisting of patient-derived spheroids (PDSp) and orthotopic xenograft models for chemotherapy and targeted therapy testing. We hypothesized that with this preclinical pipeline we could find an effective combination treatment strategy utilizing pan-histone deacetylase (HDAC) inhibition. Methods: RNA sequencing, microarray, NanoString, and immunohistochemistry (IHC) data of patient HB samples were analyzed for expression of all HDAC classes. HB cell lines were used to screen HDAC inhibitors. PDSp were used to screen which combination of standard chemotherapy regimens with a HDAC inhibitor. HB patient derived xenograft (PDX) mice models were developed and treated based off the lowest viability of PDSp drug screen. Mice were started on study when tumor volume reached 0.1-0.4 cm3 and were sacked when tumors reached diameter of 1.5 cm or at the end of the 6-week study period. Tumors were sectioned and evaluated for Ki67% and necrosis. Results: HDAC RNA and protein expression was elevated in HB tumors compared to normal livers. Four unique HB PDX models, including high risk, relapse and treatment refractory cases, were developed and validated from treatment-refractory patient tumors. Panobinostat (IC50 of 0.013-0.059 mM) showed strong in vitro effects with the lowest cell viability compared to other HDAC inhibitors. PDSp demonstrated the highest level of cell death with combination treatment of vincristine/irinotecan/panobinostat (VIP). After 6 week of treatment[SV1] , all four models had a limited response to VI therapy; however, showed a much improved response with VIP. All four models responded to therapy with a decrease in tumor size compared to placebo and survival to 6 weeks. Two of our four models that had showed some initial response to VI therapy demonstrated necrotic cell death, lower Ki67%, decreased serum alpha fetoprotein (AFP) and reduced tumor burden compared to paired vincristine/irinotecan (VI) and placebo. Conclusions: Utilizing a preclinical pipeline for HB, we have shown that panobinostat in combination with VI chemotherapy can induce an effective tumor response in models developed from high risk, relapse, and treatment refractory HB patients. This clinically relevant methodology can be used to screen novel targeted combination therapies with the VI backbone in order to create novel treatment strategies for future clinical trials. Citation Format: Andres F Espinoza, Roma Patel, Sai Govindu, Pavan Kureti, Bryan Armbruster, Sarah Woodfield, Sanjeev A Vasudevan. A novel treatment strategy for high-risk and relapse/refractory hepatoblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A003.

Four Distinct Subtypes of Alzheimer’s Disease Based on Resting-State Connectivity Biomarkers

BackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder with significant heterogeneity. Different AD phenotypes may be associated with specific brain network changes. Uncovering disease heterogeneity by using functional networks could provide insights into precise diagnoses. MethodsWe investigated the subtypes of AD using nonnegative matrix factorization clustering on the previously identified 216 resting-state functional connectivities that differed between AD and normal control subjects. We conducted the analysis using a discovery dataset (n = 809) and a validated dataset (n = 291). Next, we grouped individuals with mild cognitive impairment according to the model obtained in the AD groups. Finally, the clinical measures and brain structural characteristics were compared among the subtypes to assess their relationship with differences in the functional network. ResultsIndividuals with AD were clustered into 4 subtypes reproducibly, which included those with 1) diffuse and mild functional connectivity disruption (subtype 1), 2) predominantly decreased connectivity in the default mode network accompanied by an increase in the prefrontal circuit (subtype 2), 3) predominantly decreased connectivity in the anterior cingulate cortex accompanied by an increase in prefrontal cortex connectivity (subtype 3), and 4) predominantly decreased connectivity in the basal ganglia accompanied by an increase in prefrontal cortex connectivity (subtype 4). In addition to these differences in functional connectivity, differences between the AD subtypes were found in cognition, structural measures, and cognitive decline patterns. ConclusionsThese comprehensive results offer new insights that may advance precision medicine for AD and facilitate strategies for future clinical trials.

Addressing COVID-19 Barriers to Clinical Trial Enrollment and Implementation in the PHARM-DC Study

Recent hospitalization puts older adults at higher risk of experiencing adverse drug events (ADEs) that are a common cause of hospital readmission. Yet, most ADEs are preventable. The PHARMacist Discharge Care (PHARM-DC) study is a multi-site randomized controlled trial that seeks to evaluate the effect of pharmacist-led peri- and post-discharge interventions on 30-day hospital readmissions among older adults taking ≥10 medications or ≥3 high-risk medications. The PHARM-DC intervention includes pharmacist-led patient counseling, medication reconciliation at discharge, and a follow-up phone call post-discharge. We will highlight study protocol adaptations undertaken during the COVID-19 pandemic to address challenges to enrollment and to minimize risk of COVID-19 exposure for study participants and research personnel. Additionally, we will share insights from focus groups and semi-structured interviews with pharmacist interventionists and pharmacy leaders on barriers and facilitators to implementation due to the pandemic and strategies for future clinical trials to overcome barriers.

PD-L1 tumour expression is predictive of pazopanib response in soft tissue sarcoma

BackgroundPazopanib, a multitargeted tyrosine kinase inhibitor, is recommended as the standard treatment for refractory soft tissue sarcoma (STS). However, there are comparatively few molecular determinants for predicting pazopanib efficacy. Based on correlative studies regarding the predictive impact of PD-L1, we investigated the clinical relevance of PD-L1 expression and evaluated its value for predicting pazopanib efficacy.MethodsTumour tissues from patients with advanced STS who went on to receive pazopanib were assessed for PD-L1 expression. Immunohistochemistry was performed using an anti-PD-L1 antibody, and the PD-L1 tumour proportion score (TPS) was calculated as the percentage of at least 100 viable cells with positive expression, defined as TPS ≥ 1%.ResultsAmong the 67 patients, 8 (11.9%) achieved partial response and a median progression-free survival (PFS) of 4.8 months (95% CI 3.8–5.7). PD-L1 expression in tumour cells was detected in 13 (19.4%) cases and the TPS scores ranged from 1 to 100%, as follows: 0 (n = 54, 80.6%), 1–9% (n = 3, 4.5%), 10–49% (n = 9, 13.4%), and ≥ 50% (n = 1, 1.5%). PD-L1 positive tumours exhibited a poorer response to pazopanib treatment than the PD-L1 negative tumours (0% vs 14.8%, P = 0.07). PD-L1-positive tumours had significantly shorter PFS than the PD-L1-negative tumours (median PFS 2.8 vs 5.1 months, P = 0.003), and PD-L1 positivity was an independent predictor of poor response to pazopanib treatment (HR 2.77, 95% CI; 1.45–5.56, P = 0.006).ConclusionWe identified that PD-L1 expression can help predict the clinical outcome of patients with advanced STS treated with pazopanib. Based on our study, stratification should be actively considered in order to identify patients who will benefit from pazopanib or further therapeutic strategies for future clinical trials.

Genomic Alterations in PIK3CA-Mutated Breast Cancer Result in mTORC1 Activation and Limit the Sensitivity to PI3Kα Inhibitors

PI3Kα inhibitors have shown clinical activity in PIK3CA-mutated estrogen receptor-positive (ER+) patients with breast cancer. Using whole genome CRISPR/Cas9 sgRNA knockout screens, we identified and validated several negative regulators of mTORC1 whose loss confers resistance to PI3Kα inhibition. Among the top candidates were TSC1, TSC2, TBC1D7, AKT1S1, STK11, MARK2, PDE7A, DEPDC5, NPRL2, NPRL3, C12orf66, SZT2, and ITFG2. Loss of these genes invariably results in sustained mTOR signaling under pharmacologic inhibition of the PI3K-AKT pathway. Moreover, resistance could be prevented or overcome by mTOR inhibition, confirming the causative role of sustained mTOR activity in limiting the sensitivity to PI3Kα inhibition. Cumulatively, genomic alterations affecting these genes are identified in about 15% of PIK3CA-mutated breast tumors and appear to be mutually exclusive. This study improves our understanding of the role of mTOR signaling restoration in leading to resistance to PI3Kα inhibition and proposes therapeutic strategies to prevent or revert this resistance. SIGNIFICANCE: These findings show that genetic lesions of multiple negative regulators of mTORC1 could limit the efficacy of PI3Kα inhibitors in breast cancer, which may guide patient selection strategies for future clinical trials.

Overview of the First NRG Oncology-National Cancer Institute Workshop on Dosimetry of Systemic Radiopharmaceutical Therapy.

In 2018, the National Cancer Institute and NRG Oncology partnered for the first time to host a joint workshop on systemic radiopharmaceutical therapy (RPT) to specifically address dosimetry issues and strategies for future clinical trials. The workshop focused on current dosimetric approaches for clinical trials, strategies under development that would optimize dose reporting, and future desired or optimized approaches for novel emerging radionuclides and carriers in development. In this article, we review the main approaches that are applied clinically to calculate the absorbed dose. These include absorbed doses calculated over a variety of spatial scales, including whole body, organ, suborgan, and voxel, the last 3 of which are achievable within the MIRD schema (S value) and can be calculated with analytic methods or Monte Carlo methods, the latter in most circumstances. This article will also contrast currently available methods and tools with those used in the past, to propose a pathway whereby dosimetry helps the field by optimizing the biologic effect of the treatment and trial design in the drug approval process to reduce financial and logistical costs. We also briefly discuss the dosimetric equivalent of biomarkers to help bring a precision medicine approach to RPT implementation when merited by evidence collected during early-phase trial investigations. Advances in the methodology and related tools have made dosimetry the optimum biomarker for RPT.

Abstract ES10-3: Breast cancer patient-derived xenografts as models for metastatic progression and therapeutic response

Abstract Metastasis is the main cause of death from breast cancer, and yet little is understood with regard to how to prevent tumors from appearing in distant organs as metastases, or how to treat them once they appear. Using new models and approaches, we have discovered a new pathway regulating metastasis to lungs and bones. We found that RON kinase regulates breast cancer metastasis through cell-autonomous and non-cell-autonomous mechanisms. This presentation will cover use of various models of breast cancer to study metastasis and therapy response, including patient-derived xenograft (PDX) models. PDX models represent the diversity of human breast tumors, and can be feasibly utilized to test new drugs and drug combinations to find therapeutic approaches that match with molecular features of various tumor types. The goal of our work is to utilize PDX and other relevant in vivo models to obtain preclinical data that will facilitate prioritization of drugs to be tested in breast cancer clinical trials, including RON kinase inhibitors. Taken together, these elements converge to reveal high-priority treatment strategies for future clinical trials in breast cancer patients. Citation Format: Welm AL. Breast cancer patient-derived xenografts as models for metastatic progression and therapeutic response [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr ES10-3.

Abstract 947: CXCR4 in combination with immune check point inhibition in ovarian cancer mouse model demonstrates potential for novel therapeutic strategies

Abstract Chemokine CXCL12 and its receptor CXCR4 are the most highly expressed chemokine axis in serous ovarian cancer. CXCR4 promotes tumor growth, angiogenesis, and metastasis. High CXCR4 expression is associated with poor survival, making it an attractive therapeutic target. The objective of this study was to establish an immune competent murine ovarian cancer model with high CXCR4 expression and to test single agent CXCR4 blockade and combination therapy to optimize emerging treatment strategies for future clinical trials. CXCR4 expression was evaluated by flow cytometry in ID8, ID8-VEGF, IE9-MP1, BR5, BR5-Akt, BR5-C2k, and BR5-Kras mouse ovarian cancer cell lines as well as IE9-MP1 tumor samples. Subsequently an exploratory study was undertaken utilizing the ID8 immune competent mouse model to assess the efficacy of AMD3100, the only commercially available CXCR4 inhibitor, alone and in combination with bevacizumab and anti-PD-1. Immune analysis of circulating lymphocytes by flow cytometry was performed during treatment to monitor changes of immune cell subsets. Surface expression of CXCR4 receptor was minimal in ID8 (1.6%), ID8-VEGF (0.9%), IE9-MP1 (1.8%), BR5 (0.9%), BR5-Akt (1.2%), BR5-C2k (2.3%), and BR5-Kras (1.1%), however intracellular staining revealed widespread CXCR4 expression in ID8 (99.6%), ID8-VEGF (99.7%) BR5 (96.7%), BR5-Akt (94.3%), BR5-C2k (98.2%), and BR5-Kras (98.7%) cell lines. Using the ID8 intraperitoneal ovarian cancer mouse model in C57BL/6J mice, treatment with immunotherapy significantly increased cell surface expression of CXCR4 from 2% in the control group to 79.6-91.8% in the treatment group. Treatment with AMD3100 as a single agent showed no improvement in survival over control. However, AMD3100 in combination with anti-PD-1 therapy significantly improved survival. The addition of bevacizumab demonstrated no further survival benefit and bevacizumab as a single agent showed no improvement in survival over control, either alone or in combination with anti-PD-1. Analysis of circulating lymphocytes revealed a trend toward higher CD4 to CD8 ratio in mice who were treated with anti-PD1 therapy. Targeting the CXCL12-CXCR4 axis in combination with other immunotherapies such as immune checkpoint inhibitors is a novel therapeutic strategy which may provide benefit over single-agent immunotherapy and warrant investigation in a clinical trial. Citation Format: Kristen Starbuck, Robert McGray, Samar Masoumi-Moghaddam, Ariel Francois, Kunle Odunsi, Emese Zsiros. CXCR4 in combination with immune check point inhibition in ovarian cancer mouse model demonstrates potential for novel therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 947. doi:10.1158/1538-7445.AM2017-947

PP2A Inhibitor PME-1 Drives Kinase Inhibitor Resistance in Glioma Cells.

Glioblastoma multiforme lacks effective therapy options. Although deregulated kinase pathways are drivers of malignant progression in glioblastoma multiforme, glioma cells exhibit intrinsic resistance toward many kinase inhibitors, and the molecular basis of this resistance remains poorly understood. Here, we show that overexpression of the protein phosphatase 2A (PP2A) inhibitor protein PME-1 drives resistance of glioma cells to various multikinase inhibitors. The PME-1-elicited resistance was dependent on specific PP2A complexes and was mediated by a decrease in cytoplasmic HDAC4 activity. Importantly, both PME-1 and HDAC4 associated with human glioma progression, supporting clinical relevance of the identified mechanism. Synthetic lethality induced by both PME-1 and HDAC4 inhibition was dependent on the coexpression of proapoptotic protein BAD. Thus, PME-1-mediated PP2A inhibition is a novel mechanistic explanation for multikinase inhibitor resistance in glioma cells. Clinically, these results may inform patient stratification strategies for future clinical trials with selected kinase inhibitors in glioblastoma multiforme. Cancer Res; 76(23); 7001-11. ©2016 AACR.

Molecular Landscape of Acquired Resistance to Targeted Therapy Combinations in BRAF-Mutant Colorectal Cancer.

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. ©2016 AACR.

Emerging Therapeutic Targets for Male Germ Cell Tumors.

Male germ cell tumors (GCTs) are curable cancers, yet 10-15% of patients with metastatic disease fail cisplatin-based first-line treatments. While therapeutic options have increased for various other cancers, little progress has been made in the management of GCT in the last decades. A better understanding of the molecular alterations underlying the disease and identification of new therapeutic targets are needed. Several phase I/II studies with promising new agents are ongoing or have been completed, but most of those trials have been small and have not included translational research. Therefore, molecular profiles predictive for response or new agents have not been identified in male GCT so far. The purpose of this review is to highlight emerging targets and therapies with the potential to improve systemic treatment of metastatic male GCT and to develop strategies for future clinical trials.

Abstract B45: A pilot trial of dietary fish and ω-3 fatty acid supplements in women at high risk for breast cancer

Abstract Background: The type of fat consumed in the diet may influence mammary tissue biology and breast cancer risk, with preclinical evidence for inhibitory effects of n-3 versus n-6 polyunsaturated fatty acids (PUFAs) on mammary carcinogenesis. Fish consumption and fish oil supplements are two strategies to increase dietary n-3 fatty acids. Objective: To determine the effects of increased fish consumption on breast adipose tissue fatty acids, we conducted a study of dietary fish versus an n-3 fatty acid supplement in women at increased risk for developing breast cancer. Design: In this three month randomized study, women at high risk for breast cancer received either (a) dietary fish in the form of canned salmon and albacore tuna at a dose of four 6 ounce servings/week or (b) two n-3 fatty acid capsules (1.68 g EPA+DHA)/day for three months. Subjects underwent fasting blood draws monthly and breast adipose tissue samples at baseline and three months for fatty acid profiling by gas chromatography. Results: Both study interventions of dietary fish and n-3 PUFA capsule yielded increased EPA and DHA content in plasma (p<0.0001), erythrocyte membranes (p<0.0001), and breast adipose tissue samples (p <0.01) at 3 months, with also significantly increased total ω-3 PUFAs and decreased n-3/n-6 ratios (p<0.0001). Women taking n-3 PUFA capsules had a greater rise in plasma and erythrocyte membrane EPA relative to those consuming fish (p=0.002), without significant differences in DHA. BMI attenuated the incremental increase in EPA and DHA. Adherence was excellent in both groups at 93.9%, and higher in the dietary fish arm (p=0.01). Conclusions: Dietary fish and n-3 PUFA capsules significantly increase breast adipose tissue EPA and DHA content and represent two well tolerated strategies for future clinical trials of dietary n-3 PUFAs for breast cancer prevention. Citation Format: Shana Straka, Joanne L. Lester, Sarah Puchala, Angela Rose, Rachel Cole, Rebecca R. Andridge, Steven K. Clinton, Martha A. Belury, Lisa D. Yee. A pilot trial of dietary fish and ω-3 fatty acid supplements in women at high risk for breast cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B45.

Abstract 471: Impact of Enrichment Strategies for Future Clinical Trials Assessing PCSK-9 Inhibitors Efficacy in Primary Prevention Settings

Introduction: To date multiple ongoing clinical trials are testing impact of human monoclonal antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9) in high risk patients with established CVD already on statins. It is of interest to explore the added value of these therapies in individuals free of established CVD. The aim of this study is to assess the implications of population baseline risk considered for clinical trials, in evaluating impact of PCSK9 inhibitors in primary prevention settings. Methods: We simulated scenarios among primary prevention cohorts recruited with 5 year CVD event rates ranging from 5-25%. In the control group, 30% relative risk reduction (RRR) with highest tolerated statin dose was considered. The NNT and drug cost with additional PSCK9 inhibitor therapy to prevent one cardiac event were calculated across varying (20-40%) RRR thresholds. The annual cost of PCSK9 inhibitors was estimated in range of $3000-$7,000. We considered ≤ $0.5 million as upper limit for willingness to pay to prevent 1 event in 5 years. Results: The table highlights: a) absolute event rates anticipated in the controls as well with additional PCSK9 inhibition; b) the calculated NNT; and c) costs to prevent one cardiac event across the spectrum of baseline CVD risk levels. Among those with estimated 5 year absolute risk of 10%, the threshold for willingness to pay was achieved provided PCSK9 inhibition achieved 40% RRR with annual cost of $3000. Among individuals with >20% absolute 5 year baseline risk, the threshold for willingness to pay was realized even with 20% RRR in all scenarios if annual cost was limited to $3000. Conclusion: Future discussion for clinical trials assessing efficacy of PCSK9 inhibitors in reducing CVD events in primary prevention settings must focus on enrichment strategies for recruiting a ‘high risk’ population. These strategies will likely facilitate acceptable NNT and public willingness to pay for these emerging novel therapies.

Review of Cancer – Associated Fibroblasts and Therapies that Interfere with Their Activity

Received 09 December 2012 Accepted 17 January 2013 Abstract Cancer-associated fibroblasts (CAFs) are a specific but heterogenous population of cells that reside in the tumor microenvironment and play a significant role in the neoplastic process. CAFs participate in tumorigenesis, provide a nurturing environment for neoplastic cells, promote malignant cell growth and invasiveness by remodeling the local extracellular matrix; they have pro-inflammatory and proangiogenic properties. CAFs also confer resistance to chemotherapy and raditation therapy. In order to achieve these activities, CAFs secrete multiple growth factors, cytokines and proteins. Communication between CAFs and malignant cells is not, however, one way; premalignant cells and tumor cells secrete their own cytokines and growth factors that increase CAF numbers and function. There has also been considerable clinical research with agents that may interfere with CAFs but it has been difficult to learn about and measure the interference with CAFs per se. This review addresses the current state of knowledge about CAFs, summarizes the clinical development to date of therapeutics that inhibit CAF growth and function and suggests strategies for future clinical trials that focus particularly on CAFs and their products.

Minor clone provides a reservoir for relapse in multiple myeloma

Recent studies have provided direct evidence for genetic variegation in subclones for various cancer types. However, little is known about subclonal evolutionary processes according to treatment and subsequent relapse in multiple myeloma (MM). This issue was addressed in a cohort of 24 MM patients treated either with conventional chemotherapy or with the proteasome inhibitor, bortezomib. As MM is a highly heterogeneous disease associated with a large number of chromosomal abnormalities, a subset of secondary genetic events that seem to reflect progression, 1q21 gain, NF-κB-activating mutations, RB1 and TP53 deletions, was examined. By using high-resolution single-nucleotide polymorphism arrays, subclones were identified with nonlinear complex evolutionary histories. Such reordering of the spectrum of genetic lesions, identified in a third of MM patients during therapy, is likely to reflect the selection of genetically distinct subclones, not initially competitive against the dominant population but which survived chemotherapy, thrived and acquired new anomalies. In addition, the emergence of minor subclones at relapse appeared to be significantly associated with bortezomib treatment. These data support the idea that new strategies for future clinical trials in MM should combine targeted therapy and subpopulations' control to eradicate all myeloma subclones in order to obtain long-term remission.

Stratification of patients with neuroblastoma for targeted ALK inhibitor therapy.

9514 Background: Mutations in the ALK proto-oncogene are the major cause of hereditary neuroblastoma (NB), can be somatically acquired, and may offer a tractable therapeutic target for a subset of patients. Methods: We identified 1,604 paired constitutional and tumor DNA samples from patients representative of the natural spectrum of NB for comprehensive evaluation of ALK genomic status. This set has complete annotation of clinical covariates and patient outcome, and provides sufficient power to identify clinically relevant ALK aberrations within each NB risk group. We performed DNA sequencing of the ALK kinase domain and defined ALK copy number (CN) status using quantitative PCR. For each tumor where a somatic mutation was identified, we sequenced matched germline DNA, allowing us to understand the frequency of hereditary predisposition to NB. Results: Mutations were discovered in 126/1,604 (7.9%) primary NB tumor samples distributed across the spectrum of phenotypes. Mutations at R1275 were most common (n=54) followed by F1174 (n=39) and F1245 (n=15), with those 3 locations accounting for 86% of all mutations. I1170 and I1171 were identified twice and 17 other previously unidentified mutations were seen only once. Work is ongoing to determine the functional significance of these mutations, as is evaluation of matched germline DNA. We found a statistically significant higher number of ALK mutations in high-risk versus low-risk cases (p=0.018) and in cases with MYCN amplification (p=0.031). There was no significant difference between other risk stratification covariates including age (< or > 1 year, p=0.066), histology (p=0.103) and ploidy (0.922). ALK CN gain was found in 197/1345 (14.6%) samples, while those with amplification were identified in 24/1345 (1.8%) and almost exclusively seen concurrently with MYCN amplification. CN gain was seen more frequently in high-risk, as compared with low- and intermediate-risk, disease (p<0.01). Conclusions: Combining ALK mutation and CN data overall and within each risk group and correlating this with other genetic factors, clinical phenotype, and patient outcome will allow us to determine the clinical significance of ALK mutations in NB and develop strategies for future clinical trials.

Integrated molecular analysis suggests a three-class model for low-grade gliomas: A proof-of-concept study

Introduction: We used an integrated molecular analysis strategy to perform class discovery on a population of low-grade gliomas (astrocytomas, oligodendrogliomas, and mixed gliomas) to improve our understanding of the molecular relationships among these tumors and to reconcile genotypic relationships with current histologic and molecular strategies for tumor classification. Methods: Gene expression profiling was performed on a cross-section of World Health Organization (WHO) grades I–II gliomas. Unsupervised class discovery algorithms identified and validated tumor clusters with genotypic similarity, and these data were integrated with chromosomal copy number assays and RT-PCR data to define molecular tumor subclasses. Machine learning models allowed accurate, prospective classification of unknown tumors into these molecular subgroups. This molecular classification model was compared to current histologic (WHO) and molecular pathologic (chromosome 1p and 19q deletions, p53 alterations, and Ki-67 expression) methods for glioma classification. Results: Molecular class discovery suggested a three-class model for low-grade gliomas. One discrete cluster of gliomas identified the pilocytic astrocytomas, a second grouped the 1p/19q codeleted oligodendrogliomas, and the mixture of remaining 1p/19q intact gliomas, including astrocytomas, oligodendrogliomas, and oligoastrocytomas, formed a third cluster with a discrete pattern of expression. Conclusions: Integration of genomic, transcriptomic, and morphologic data for class discovery suggests a three-class model for low-grade gliomas. Class I represents tumors with molecular similarity to pilocytic astrocytomas, class II tumors are similar to 1p/19q codeleted oligodendrogliomas, and class III represents infiltrative low-grade gliomas. This classification is similar to current clinical paradigms for low-grade gliomas; our work suggests a molecular basis for such models. This classification may supplement or may serve as the basis for a molecular pathologic alternative to current grading schemes for low-grade gliomas and may highlight potential targets for future biologically based treatments or strategies for future clinical trials.

Rapamycin weekly maintenance dosing and the potential efficacy of combination sorafenib plus rapamycin but not atorvastatin or doxycycline in tuberous sclerosis preclinical models

BackgroundTuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome, characterized by hamartomatous growths in the brain, skin, kidneys, lungs, and heart, which lead to significant morbidity. TSC is caused by mutations in the TSC1 or TSC2 genes, whose products, hamartin and tuberin, form a tumor suppressor complex that regulates the PI3K/Akt/mTOR pathway. Early clinical trials show that TSC-related kidney tumors (angiomyolipomas) regress when treated with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin (also known as sirolimus). Although side effects are tolerable, responses are incomplete, and tumor regrowth is common when rapamycin is stopped. Strategies for future clinical trials may include the investigation of longer treatment duration and combination therapy of other effective drug classes.ResultsHere, we examine the efficacy of a prolonged maintenance dose of rapamycin in Tsc2+/- mice with TSC-related kidney tumors. Cohorts were treated with rapamycin alone or in combination with interferon-gamma (IFN-g). The schedule of rapamycin included one month of daily doses before and after five months of weekly doses. We observed a 94.5% reduction in kidney tumor burden in Tsc2+/- mice treated (part one) daily with rapamycin (8 mg/kg) at 6 months ≤ age < 7 months, (part 2) weekly with rapamycin (16 mg/kg) at 7 months ≤ age < 12 months, and (part 3) daily with rapamycin (8 mg/kg) at 12 months ≤ age < 13 months; but we did not observe any improvement with combination IFN-g plus rapamycin in this study. We also used a Tsc2-/- subcutaneous tumor model to evaluate other classes of drugs including sorafenib, atorvastatin, and doxycycline. These drugs were tested as single agents and in combination with rapamycin. Our results demonstrate that the combination of rapamycin and sorafenib increased survival and may decrease tumor volume as compared to rapamycin treatment alone while sorafenib as a single agent was no different than control. Atorvastatin and doxycycline, either as single agents or in combination with rapamycin, did not improve outcomes as compared with controls.ConclusionOur results indicate that prolonged treatment with low doses of mTOR inhibitors may result in more complete and durable TSC-related tumor responses, and it would be reasonable to evaluate this strategy in a clinical trial. Targeting the Raf/Mek/Erk and/or VEGF pathways in combination with inhibiting the mTOR pathway may be another useful strategy for the treatment of TSC-related tumors.

Postoperative Chemotherapy in Resected Gastric Cancer: Results of a Single Center Experience

Gastric cancer remains a major health problem despite its decline in incidence in Western countries. Although radical surgery represents the primary curative option for gastric cancer patients, most of them relapse and die due to their disease despite an R0 resection. At present the routine use of postoperative adjuvant therapy to reduce disease recurrence is still considered an investigational approach. Out of a total of 275 patients (stage IB through IV M0 AJCC/UICC) who underwent surgery for gastric cancer at our Surgery Unit between 1993 and 2001, 156 were eligible for adjuvant chemotherapy, of whom only 52 accepted to undergo this treatment. This group of patients was retrospectively compared with a control group (1:2) and overall survival was assessed using hazard ratio and Kaplan-Meier estimates. Five-year survival was 40% in the chemotherapy group and 37.8% in the group which underwent surgery alone. Indeed, chemotherapy did not reduce the risk of death (HR 0.87, 95% CI = 0.57-1.34, p=0.54). Serosal involvement and the invasion of more than 6 lymph nodes were the main independent prognostic factors identified by multivariate analysis. The current study did not show a clear advantage of chemotherapy over surgery alone. However, our results can help to define strategies for future clinical trials with the use of new regimens based on more effective and less toxic drugs.