PRAME immunohistochemistry is useful in the evaluation of conjunctival melanomas, nevi, and primary acquired melanosis.

BACKGROUND: Many dermatopathologists find conjunctival melanocytic proliferations challenging because they are rare relative to their cutaneous counterparts and have nuanced morphology and nomenclature. PRAME immunohistochemistry has been widely adopted for distinguishing cutaneous nevi from melanoma, but limited data exist assessing its utility in evaluating conjunctival specimens. In particular, it is uncertain whether it can predict the risk of melanoma progression in primary acquired melanosis (PAM). METHODS: Thirty clinically annotated cases (two melanomas, three PAM with atypia, seven PAM without atypia, 15 nevi, two combined nevi, and a diagnostically challenging nevus with atypical features) were retrospectively evaluated with PRAME. RESULTS: Strong, diffuse PRAME expression was present in melanomas and PAM with high-grade atypia, but not in PAM with low-grade atypia, PAM without atypia, or nevi. Scattered, faintly PRAME-positive intraepithelial melanocyte nuclei were identified in six nevi. A clonal nevus and nests of heavily pigmented type-A melanocytes in two additional nevi had cytoplasmic staining. CONCLUSIONS: PRAME was useful for distinguishing melanoma and its probable precursors from benign conjunctival melanocytic proliferations in our cohort. The data alert us to two diagnostic pitfalls in nevi: scattered, PRAME-positive intraepithelial melanocytes and cytoplasmic PRAME staining in type-A melanocytes and melanophages. Larger scale investigations are warranted to further substantiate these promising findings.

A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine.

Nucleolin Overexpression Confers Increased Sensitivity to the Anti-Nucleolin Aptamer, AS1411.

AS1411 is an antiproliferative DNA aptamer, which binds the ubiquitous protein, nucleolin. In this study, we show that constitutive overexpression of nucleolin confers increased sensitivity to the growth inhibitory effects of AS1411. HeLa cells overexpressing nucleolin have an increased growth rate and invasiveness relative to control cells. Nucleolin overexpressing cells demonstrate increased growth inhibition in response to the AS1411 treatment, which correlates with increased apoptosis and cell cycle arrest, when compared to non-transfected cells. AS1411 induces nucleolin expression at the RNA and protein level in HeLa cells, suggesting a feedback loop with important implications for the clinical use of AS1411.

c-Myc quadruplex-forming sequence Pu-27 induces extensive damage in both telomeric and nontelomeric regions of DNA.

Quadruplex-forming DNA sequences are present throughout the eukaryotic genome, including in telomeric DNA. We have shown that the c-Myc promoter quadruplex-forming sequence Pu-27 selectively kills transformed cells (Sedoris, K. C., Thomas, S. D., Clarkson, C. R., Muench, D., Islam, A., Singh, R., and Miller, D. M. (2012) Genomic c-Myc quadruplex DNA selectively kills leukemia. Mol. Cancer Ther. 11, 66-76). In this study, we show that Pu-27 induces profound DNA damage, resulting in striking chromosomal abnormalities in the form of chromatid or chromosomal breaks, radial formation, and telomeric DNA loss, which induces γ-H2AX in U937 cells. Pu-27 down-regulates telomeric shelterin proteins, DNA damage response mediators (RAD17 and RAD50), double-stranded break repair molecule 53BP1, G2 checkpoint regulators (CHK1 and CHK2), and anti-apoptosis gene survivin. Interestingly, there are no changes of DNA repair molecules H2AX, BRCA1, and the telomere maintenance gene, hTERT. ΔB-U937, where U937 cells stably transfected with deleted basic domain of TRF2 is partially sensitive to Pu-27 but exhibits no changes in expression of shelterin proteins. However, there is an up-regulation of CHK1, CHK2, H2AX, BRCA1, and survivin. Telomere dysfunction-induced foci assay revealed co-association of TRF1with γ-H2AX in ATM deficient cells, which are differentially sensitive to Pu-27 than ATM proficient cells. Alt (alternating lengthening of telomere) cells are relatively resistant to Pu-27, but there are no significant changes of telomerase activity in both Alt and non-Alt cells. Lastly, we show that this Pu-27-mediated sensitivity is p53-independent. The data therefore support two conclusions. First, Pu-27 induces DNA damage within both telomeric and nontelomeric regions of the genome. Second, Pu-27-mediated telomeric damage is due, at least in part, to compromise of the telomeric shelterin protein complex.

gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma.

BACKGROUND: Stimulating an immune response against cancer with the use of vaccines remains a challenge. We hypothesized that combining a melanoma vaccine with interleukin-2, an immune activating agent, could improve outcomes. In a previous phase 2 study, patients with metastatic melanoma receiving high-dose interleukin-2 plus the gp100:209-217(210M) peptide vaccine had a higher rate of response than the rate that is expected among patients who are treated with interleukin-2 alone. METHODS: We conducted a randomized, phase 3 trial involving 185 patients at 21 centers. Eligibility criteria included stage IV or locally advanced stage III cutaneous melanoma, expression of HLA*A0201, an absence of brain metastases, and suitability for high-dose interleukin-2 therapy. Patients were randomly assigned to receive interleukin-2 alone (720,000 IU per kilogram of body weight per dose) or gp100:209-217(210M) plus incomplete Freund's adjuvant (Montanide ISA-51) once per cycle, followed by interleukin-2. The primary end point was clinical response. Secondary end points included toxic effects and progression-free survival. RESULTS: The treatment groups were well balanced with respect to baseline characteristics and received a similar amount of interleukin-2 per cycle. The toxic effects were consistent with those expected with interleukin-2 therapy. The vaccine-interleukin-2 group, as compared with the interleukin-2-only group, had a significant improvement in centrally verified overall clinical response (16% vs. 6%, P=0.03), as well as longer progression-free survival (2.2 months; 95% confidence interval [CI], 1.7 to 3.9 vs. 1.6 months; 95% CI, 1.5 to 1.8; P=0.008). The median overall survival was also longer in the vaccine-interleukin-2 group than in the interleukin-2-only group (17.8 months; 95% CI, 11.9 to 25.8 vs. 11.1 months; 95% CI, 8.7 to 16.3; P=0.06). CONCLUSIONS: In patients with advanced melanoma, the response rate was higher and progression-free survival longer with vaccine and interleukin-2 than with interleukin-2 alone. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00019682.).

Airway management evolution - in a search for an ideal extraglottic airway device.

Extraglottic airway devices (EADs) are commonly used equipment for airway maintenance during elective procedures under general anaesthesia. They may be used also in other indications such as conduit for tracheal intubation or rescue airway device in prehospital medicine. Current classifications of the EADs lack systematic approach and therefore classification according to the sealing sites and sealing mechanisms is suggested in this review article. Modern EADs are disposable, latex-free devices made of plastic materials most commonly from polyvinylchloride (PVC). The bowl of uncuffed sealers is manufactured from different materials such as thermoplastic elastomers or ethylene-vinyl-acetate co-polymer. EADs create various physical forces exerted on the adjacent tissues which may contribute to different sealing characteristic of particular device or to variable incidence of postoperative complications. Desired features of an ideal EAD involve easy insertion, high insertion success rate even by inexperienced users, protection against aspiration of gastric contents and low incidence of postoperative complications such as sore throat, hoarseness, cough or swallowing difficulties.

Spatially Resolved Microglia/Macrophages in Recurrent Glioblastomas Overexpress Fatty Acid Metabolism and Phagocytic Genes.

BACKGROUND: Glioblastoma (GBM) tumors are rich in tumor-associated microglia/macrophages. Changes associated with treatment in this specific cell population are poorly understood. Therefore, we studied changes in gene expression of tumor-associated microglia/macrophages (Iba1+) cells in de novo versus recurrent GBMs. METHODS: NanoString GeoMx® Digital Spatial Transcriptomic Profiling of microglia/macrophages (Iba1+) and glial cells (Gfap+) cells identified on tumor sections was performed on paired de novo and recurrent samples obtained from three IDH-wildtype GBM patients. The impact of differentially expressed genes on patient survival was evaluated using publicly available data. RESULTS: Unsupervised analyses of the NanoString GeoMx® Digital Spatial Profiling data revealed clustering based on the transcriptomic data from Iba1+ and Gfap+ cells. As expected, conventional differential gene expression and enrichment analyses revealed upregulation of immune-function-related genes in Iba1+ cells compared to Gfap+ cells. A focused differential gene expression analysis revealed upregulation of phagocytosis and fatty acid/lipid metabolism genes in Iba1+ cells in recurrent GBM samples compared to de novo GBM samples. Importantly, of these genes, the lipid metabolism gene PLD3 consistently correlated with survival in multiple different publicly available datasets. CONCLUSION: Tumor-associated microglia/macrophages in recurrent GBM overexpress genes involved in fatty acid/lipid metabolism. Further investigation is needed to fully delineate the role of PLD phospholipases in GBM progression.

Tumor-associated primo vascular system is derived from xenograft, not host.

The primo vascular system (PVS), which is composed of very small primo-vessels (PV) and primo-nodes (PN), has recently emerged as a third component of circulatory system. Here, we report the presence of a tumor derived PVS in murine xenografts of human histiocytic lymphoma (U937) in close proximity to the tumor. Within this system, PNs are small (~500-600 µM diameter) membranous sac-like structures which contain numerous small cells which can be demonstrated by DAPI staining. Hematoxylin and Eosin (H&E) staining of the peri-tumoral PVS shows the presence of loose structures lined by fibroblasts but filled with dense fibers, cells, lacunae and nerve-like structures. The origin and type of cells within the PVS was characterized by immunostaining with antibodies for CD68, CD45 and lysozyme. The results of these studies reveal that the PVS of the xenograft originates from the human U937 tumor cells. qRT-PCR analysis of mRNA isolated from PVS cells reveals a striking predominance of human, rather than mouse, sequences. Of particular interest, human stem cell specific transcription factors were overexpressed, most notably KLF4, an upstream regulator of NANOG which maintains the pluripotent and undifferentiated state of stem cells. These results suggest that the cells present within the PVS are derived from the human xenograft and suggests that the primo-vessels associated with the xenografted tumor may provide a safe haven for a select population of cancer stem cells. Further understanding of the biological properties of these cells may allow the development of new anti-cancer interventions.

Evaluation of Lung Cancer Patient Response to First-Line Chemotherapy by Integration of Tumor Core Biopsy Metabolomics with Multiscale Modeling.

The standard of care for intermediate (Stage II) and advanced (Stages III and IV) non-small cell lung cancer (NSCLC) involves chemotherapy with taxane/platinum derivatives, with or without radiation. Ideally, patients would be screened a priori to allow non-responders to be initially treated with second-line therapies. This evaluation is non-trivial, however, since tumors behave as complex multiscale systems. To address this need, this study employs a multiscale modeling approach to evaluate first-line chemotherapy response of individual patient tumors based on metabolomic analysis of tumor core biopsies obtained during routine clinical evaluation. Model parameters were calculated for a patient cohort as a function of these metabolomic profiles, previously obtained from high-resolution 2DLC-MS/MS analysis. Evaluation metrics were defined to classify patients as Disease-Control (DC) [encompassing complete-response (CR), partial-response (PR), and stable-disease (SD)] and Progressive-Disease (PD) following first-line chemotherapy. Response was simulated for each patient and compared to actual response. The results show that patient classifications were significantly separated from each other, and also when grouped as DC vs. PD and as CR/PR vs. SD/PD, by fraction of initial tumor radius metric at 6 days post simulated bolus drug injection. This study shows that patient first-line chemotherapy response can in principle be evaluated from multiscale modeling integrated with tumor tissue metabolomic data, offering a first step towards individualized lung cancer treatment prognosis.

Plasma Thermogram Parameters Differentiate Status and Overall Survival of Melanoma Patients.

Melanoma is the fifth most common cancer in the United States and the deadliest of all skin cancers. Even with recent advancements in treatment, there is still a 13% two-year recurrence rate, with approximately 30% of recurrences being distant metastases. Identifying patients at high risk for recurrence or advanced disease is critical for optimal clinical decision-making. Currently, there is substantial variability in the selection of screening tests and imaging, with most modalities characterized by relatively low accuracy. In the current study, we built upon a preliminary examination of differential scanning calorimetry (DSC) in the melanoma setting to examine its utility for diagnostic and prognostic assessment. Using regression analysis, we found that selected DSC profile (thermogram) parameters were useful for differentiation between melanoma patients and healthy controls, with more complex models distinguishing melanoma patients with no evidence of disease from patients with active disease. Thermogram features contributing to the third principal component (PC3) were useful for differentiation between controls and melanoma patients, and Cox proportional hazards regression analysis indicated that PC3 was useful for predicting the overall survival of active melanoma patients. With the further development and optimization of the classification method, DSC could complement current diagnostic strategies to improve screening, diagnosis, and prognosis of melanoma patients.

Untangling the web of glioblastoma treatment resistance using a multi-omic and multidisciplinary approach.

Glioblastoma (GBM), the most common human brain tumor, has been notoriously resistant to treatment. As a result, the dismal overall survival of GBM patients has not changed over the past three decades. GBM has been stubbornly resistant to checkpoint inhibitor immunotherapies, which have been remarkably effective in the treatment of other tumors. It is clear that GBM resistance to therapy is multifactorial. Although therapeutic transport into brain tumors is inhibited by the blood brain barrier, there is evolving evidence that overcoming this barrier is not the predominant factor. GBMs generally have a low mutation burden, exist in an immunosuppressed environment and they are inherently resistant to immune stimulation, all of which contribute to treatment resistance. In this review, we evaluate the contribution of multi-omic approaches (genomic and metabolomic) along with analyzing immune cell populations and tumor biophysical characteristics to better understand and overcome GBM multifactorial resistance to treatment.

Malignant melanotic nerve sheath tumor with PRKAR1A, KMT2C, and GNAQ mutations.

Malignant melanotic nerve sheath tumor (MMNST) is a rare and potentially aggressive lesion defined in the 2021 WHO Classification of Tumors of the Central Nervous System. MMNST demonstrate overlapping histologic and clinical features of schwannoma and melanoma. MMNST often harbor PRKAR1A mutations, especially within the Carney Complex. We present a case of aggressive MMNST of the sacral region in a 48-year-old woman. The tumor contained PRKAR1A frameshift pR352Hfs*89, KMT2C splice site c.7443-1G>T and GNAQ p.R183L missense mutations, as well as BRAF and MYC gains. Genomic DNA methylation analysis using the Illumina 850K EpicBead chip revealed that the lesion did not match an established methylation class; however, uniform manifold approximation and projection (UMAP) placed the tumor very near schwannomas. The tumor expressed PD-L1, and the patient was treated with radiation and immune checkpoint inhibitors following en bloc resection. Although she had symptomatic improvement, she suffered early disease progression with local recurrence, and distant metastases, and died 18 months after resection. It has been suggested that the presence of GNAQ mutations can differentiate leptomeningeal melanocytic neoplasms and uveal melanoma from MMNST. This case and others demonstrate that GNAQ mutations may exist in malignant nerve sheath tumors; that GNAQ and PRKAR1A mutations are not always mutually exclusive and that neither can be used to differentiate MMNST or MPNST from all melanocytic lesions.

Phase II trial of the regulatory T cell-depleting agent, denileukin diftitox, in patients with unresectable stage IV melanoma.

BACKGROUND: We previously found that administration of an interleukin 2/diphtheria toxin conjugate (DAB/IL2; Denileukin Diftitox; ONTAK) to stage IV melanoma patients depleted CD4(+)CD25(HI)Foxp3(+) regulatory T cells and expanded melanoma-specific CD8(+) T cells. The goal of this study was to assess the clinical efficacy of DAB/IL2 in an expanded cohort of stage IV melanoma patients. METHODS: In a single-center, phase II trial, DAB/IL2 (12 µg/kg; 4 daily doses; 21 day cycles) was administered to 60 unresectable stage IV melanoma patients and response rates were assessed using a combination of 2-[(18)F]-fluoro-2-deoxy-glucose (FDG)-positron emission tomography (PET) and computed tomography (CT) imaging. RESULTS: After DAB/IL2 administration, 16.7% of the 60 patients had partial responses, 5% stable disease and 15% mixed responses. Importantly, 45.5% of the chemo/immuno-naïve sub-population (11/60 patients) experienced partial responses. One year survival was markedly higher in partial responders (80 ± 11.9%) relative to patients with progressive disease (23.7 ± 6.5%; p value < 0.001) and 40 ± 6.2% of the total DAB/IL2-treated population were alive at 1 year. CONCLUSIONS: These data support the development of multi-center, randomized trials of DAB/IL2 as a monotherapy and in combination with other immunotherapeutic agents for the treatment of stage IV melanoma. TRIAL REGISTRATION: NCT00299689.

Bioengineered Models to Study Microenvironmental Regulation of Glioblastoma Metabolism.

Despite extensive research and aggressive therapies, glioblastoma (GBM) remains a central nervous system malignancy with poor prognosis. The varied histopathology of GBM suggests a landscape of differing microenvironments and clonal expansions, which may influence metabolism, driving tumor progression. Indeed, GBM metabolic plasticity in response to differing nutrient supply within these microenvironments has emerged as a key driver of aggressiveness. Additionally, emergent biophysical and biochemical interactions in the tumor microenvironment (TME) are offering new perspectives on GBM metabolism. Perivascular and hypoxic niches exert crucial roles in tumor maintenance and progression, facilitating metabolic relationships between stromal and tumor cells. Alterations in extracellular matrix and its biophysical characteristics, such as rigidity and topography, regulate GBM metabolism through mechanotransductive mechanisms. This review highlights insights gained from deployment of bioengineering models, including engineered cell culture and mathematical models, to study the microenvironmental regulation of GBM metabolism. Bioengineered approaches building upon histopathology measurements may uncover potential therapeutic strategies that target both TME-dependent mechanotransductive and biomolecular drivers of metabolism to tackle this challenging disease. Longer term, a concerted effort integrating in vitro and in silico models predictive of patient therapy response may offer a powerful advance toward tailoring of treatment to patient-specific GBM characteristics.

Evaluation of disease staging and chemotherapeutic response in non-small cell lung cancer from patient tumor-derived metabolomic data.

OBJECTIVES: Despite extensive effort, the search for clinically-relevant metabolite biomarkers for early detection, disease monitoring, and outcome prediction in lung cancer remains unfulfilled. Although biofluid evaluation has been explored, the complexity inherent in metabolite data and the dynamic discrepancy between metabolites in biofluids vs. tumor tissue have prevented conclusive results. This proof-of-concept study explored models predictive of staging and chemotherapy response based on metabolomic analysis of fresh, patient-derived non-small cell lung cancer (NSCLC) core biopsies. MATERIALS AND METHODS: Samples (n = 36) were evaluated with high-resolution 2DLC-MS/MS and 13C-glucose enrichment, and the data were comprehensively analyzed with machine learning techniques. Patients were categorized as Disease-Control (DC) [encompassing complete-response (CR), partial-response (PR), and stable-disease (SD)] and Progressive-Disease (PD) in terms of first-line chemotherapy. Four major types of learning methods (partial least squares discriminant analysis (PLS-DA), support vector machines (SVM), artificial neural networks, and random forests (RF)) were applied to differentiate between positive (DC and CR/PR) and poor (PD and SD/PD) responses, and between stage I/II/III and stage IV disease. Models were trained with forward feature selection based on variable importance and tested on validation subsets. RESULTS: The models predicted patient classifications in the validation subsets with AUC (95 % CI): DC vs. PD (SVM), 0.970(0.961-0.979); CR/PR vs. SD/PD (PLS-DA), 0.880(0.865-0.895); stage I/II/III vs. IV (SVM), 0.902(0.880-0.924). Highest performing model was SVM for DC vs. PD (balanced accuracy = 0.92; kappa = 0.74). CONCLUSION: This study illustrates a comprehensive evaluation of patient tumor-specific metabolic profiles, with the potential to identify disease stage and predict response to first-line chemotherapy.

Hypoxia induces differential translation of enolase/MBP-1.

BACKGROUND: Hypoxic microenvironments in tumors contribute to transformation, which may alter metabolism, growth, and therapeutic responsiveness. The alpha-enolase gene encodes both a glycolytic enzyme (alpha-enolase) and a DNA-binding tumor suppressor protein, c-myc binding protein (MBP-1). These divergent alpha-enolase gene products play central roles in glucose metabolism and growth regulation and their differential regulation may be critical for tumor adaptation to hypoxia. We have previously shown that MBP-1 and its binding to the c-myc P2 promoter regulates the metabolic and cellular growth changes that occur in response to altered exogenous glucose concentrations. RESULTS: To examine the regulation of alpha-enolase and MBP-1 by a hypoxic microenvironment in breast cancer, MCF-7 cells were grown in low, physiologic, or high glucose under 1% oxygen. Our results demonstrate that adaptation to hypoxia involves attenuation of MBP-1 translation and loss of MBP-1-mediated regulation of c-myc transcription, evidenced by decreased MBP-1 binding to the c-myc P2 promoter. This allows for a robust increase in c-myc expression, "early c-myc response", which stimulates aerobic glycolysis resulting in tumor acclimation to oxidative stress. Increased alpha-enolase mRNA and preferential translation/post-translational modification may also allow for acclimatization to low oxygen, particularly under low glucose concentrations. CONCLUSIONS: These results demonstrate that malignant cells adapt to hypoxia by modulating alpha-enolase/MBP-1 levels and suggest a mechanism for tumor cell induction of the hyperglycolytic state. This important "feedback" mechanism may help transformed cells to escape the apoptotic cascade, allowing for survival during limited glucose and oxygen availability.

Altered regulation of metabolic pathways in human lung cancer discerned by (13)C stable isotope-resolved metabolomics (SIRM).

BACKGROUND: Metabolic perturbations arising from malignant transformation have not been systematically characterized in human lung cancers in situ. Stable isotope resolved metabolomic analysis (SIRM) enables functional analysis of gene dysregulations in lung cancer. To this purpose, metabolic changes were investigated by infusing uniformly labeled 13C-glucose into human lung cancer patients, followed by resection and processing of paired non-cancerous lung and non small cell carcinoma tissues. NMR and GC-MS were used for 13C-isotopomer-based metabolomic analysis of the extracts of tissues and blood plasma. RESULTS: Many primary metabolites were consistently found at higher levels in lung cancer tissues than their surrounding non-cancerous tissues. 13C-enrichment in lactate, Ala, succinate, Glu, Asp, and citrate was also higher in the tumors, suggesting more active glycolysis and Krebs cycle in the tumor tissues. Particularly notable were the enhanced production of the Asp isotopomer with three 13C-labeled carbons and the buildup of 13C-2,3-Glu isotopomer in lung tumor tissues. This is consistent with the transformations of glucose into Asp or Glu via glycolysis, anaplerotic pyruvate carboxylation (PC), and the Krebs cycle. PC activation in tumor tissues was also shown by an increased level of pyruvate carboxylase mRNA and protein. CONCLUSION: PC activation - revealed here for the first time in human subjects - may be important for replenishing the Krebs cycle intermediates which can be diverted to lipid, protein, and nucleic acid biosynthesis to fulfill the high anabolic demands for growth in lung tumor tissues. We hypothesize that this is an important event in non-small cell lung cancer and possibly in other tumor development.

Prospects for clinical cancer metabolomics using stable isotope tracers.

Metabolomics provides a readout of the state of metabolism in cells or tissue and their responses to external perturbations. For this reason, the approach has great potential in clinical diagnostics. For more than two decades, we have been using stable isotope tracer approaches to probe cellular metabolism in greater detail. The ability to enrich common compounds with rare isotopes such as carbon ((13)C) and nitrogen ((15)N) is the only practical means by which metabolic pathways can be traced, which entails following the fate of individual atoms from the source molecule to products via metabolic transformation. Changes in regulation of pathways are therefore captured by this approach, which leads to deeper understanding of the fundamental biochemistry of cells. Using lessons learned from pathways tracing in cells and organs, we have been applying this methodology to human cancer patients in a clinical setting. Here we review the methodologies and approaches to stable isotope tracing in cells, animal models and in humans subjects.

Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer.

Certain guanine-rich (G-rich) DNA and RNA molecules can associate intermolecularly or intramolecularly to form four stranded or "quadruplex" structures, which have unusual biophysical and biological properties. Several synthetic G-rich quadruplex-forming oligodeoxynucleotides have recently been investigated as therapeutic agents for various human diseases. We refer to these biologically active G-rich oligonucleotides as aptamers because their activities arise from binding to protein targets via shape-specific recognition (analogous to antibody-antigen binding). As therapeutic agents, the G-rich aptamers may have some advantages over monoclonal antibodies and other oligonucleotide-based approaches. For example, quadruplex oligonucleotides are non-immunogenic, heat stable and they have increased resistance to serum nucleases and enhanced cellular uptake compared to unstructured sequences. In this review, we describe the characteristics and activities of G-rich oligonucleotides. We also give a personal perspective on the discovery and development of AS1411, an antiproliferative G-rich phosphodiester oligonucleotide that is currently being tested as an anticancer agent in Phase II clinical trials. This molecule functions as an aptamer to nucleolin, a multifunctional protein that is highly expressed by cancer cells, both intracellularly and on the cell surface. Thus, the serendipitous discovery of the G-rich oligonucleotides also led to the identification of nucleolin as a new molecular target for cancer therapy.

Human papillomavirus in metastatic squamous carcinoma from unknown primaries in the head and neck: a retrospective 7 year study.

GOAL: Human Papillomavirus (HPV) is found to be increasingly implicated in head and neck cancers. The objective of this study was to determine the primary site of origin of HPV positive squamous carcinomas metastatic to lymph nodes of the neck. METHODS: Surgical pathology records from January 1, 2000 to July 31, 2007 were used to identify surgically removed neck lymph nodes with the diagnosis of metastatic squamous carcinoma. Specimens in formalin-fixed, paraffin-embedded blocks were examined for HPV (+) by analyzing sequencing data generated by PCR and immunostaining for the expression of the p16INK biomarker, which is overexpressed if Rb is not present. H & E stained slides were also reviewed for histological classification. The available retrospective demographics were extracted from the charts to determine trends of confounding factors. RESULTS: Of the 43 patient samples, 41 contained adequate DNA to test for HPV. The mean age of the 41 patients was 62 years. All of the patients smoked and 39/41 patients consumed alcohol. The overall HPV (+) incident rate was 27% (11/41) by PCR with strongly diffuse or strong focal p16 staining. 9 of the 34 males and 2 of the 7 females had HPV (+) carcinomas. The average age of the 2 HPV (+) females was 44, compared to the HPV (-) females who averaged 70. The average age of the HPV (+) males was 56 compared with the average age 55 of the HPV (-) males. HPV (+) carcinomas appeared to arise from multiple sites in the oropharynx, particularly the tonsils and tongues, including unknown primaries. By histological exam, most metastatic HPV(+) squamous carcinomas were poorly differentiated (basaloid) microscopically and grossly cystic. CONCLUSION: The 27% HPV (+) squamous cancers metastatic to neck lymph node originated from multiple sites in the oropharynx. The HPV (+) female population, although a total of only 2, tended to be much younger than the HPV (-) ones, whereas the HPV (+) male population was similar in age to the HPV (-) male population.