The aim of the Molecular Therapeutics Unit (MTU) is the identification of novel biochemical pathways altered in HNSCC leading to the discovery of small molecules, that may modulate molecular events important for oral carcinogenesis, and thus may represent novel chemotherapeutic agents. Furthermore, the unit focuses in the preclinical assessment of biochemical and molecular parameters modulated by these small molecules, which may help to monitor the effects on tumor samples in patients with head and neck cancer receiving these novel treatment modalities. Finally, selected signaling agents developed in the unit are tested in early clinical trials in patients with advanced HNSCC and other malignancies for proof of principle testing, in order to assess whether the preclinical effects observed occur also in humans; novel effects observed in human trials are then, investigated in our unit, reverse translation. Molecular and biochemical effects of small molecules cell cycle modulators in patients with advanced neoplasms Although preclinical toxicological studies often predict human toxicity, many unwanted drug effects occur only in humans. Preclinical toxicity studies in animals suggested that flavopiridol had anti-inflammatory effects and that neutropenia would be the main toxicity. However, the dose limiting toxicity in humans was secretory diarrhea and a pro-inflammatory syndrome. The pro-inflammatory syndrome consists of fever, fatigue, and local tumor pain with concomitant alterations in plasma acute-phase reactant proteins (i.e. CRP, fibrinogen). It is quite devastating, precluding chronic therapy with this agent. To determine which cytokine was the responsible for flavopiridols effects, we tested a battery of cytokines (such as interleukin (IL)-6, IL-10, IL-12, TNF alpha levels by standard ELISA) in serial plasma samples drawn preinfusion and during the infusion. There was a significant and sustained increase in plasma IL-6 levels at all time points when compared with baseline values. The relative increase in IL-6 concentration was dose-dependent. Moreover, IL-6 elevation had a direct correlation with flavopiridol peak plasma concentration, flavopiridol area under the curve, and plasma C-Reactive protein levels. No changes in the other pro or anti-inflammatory cytokines were observed. Immunohistochemistry studies (IHC) in bone marrow aspirates from a prospective group of patients in this trial demonstrated approximately 4-fold induction of IL-6 (compared with baseline), mostly in non-T cells. The mechanism(s) underlying IL-6 induction and its significance are still unknown but may influence strategies to modulate flavopiridols clinical effects. These efforts were published recently. Initial mechanistic studies in our unit revealed that the accumulation of cytokines by flavopiridol may be due to activation of NF-kappa beta and the p38 MAPK pathway. Studies using inhibitors of these pathways are being tested in in vitro and in vivo models. This information will help us in ameliorating this side effect, leading to a better therapeutic index of this agent. Future phase I trials using flavopiridol with cytokine modulators in patients with advanced neoplasms are being considered in our unit. We predict that inhibitors of pro-inflammatory cytokines may increase the therapeutic index of this agent. Based on this information we are in the process of writing a Phase I trial of flavopiridol in combination with inhibitors of cytokine production to be conducted at the CC in order to test this hypothesis. Searching for available tissues (accessible by non-invasive means), we hypothesized that oral keratinocytes obtained by oral cytobrush may be a good source (surrogate tissue) to study drug effects (surrogate markers). After significant efforts in our lab we are now able to assess the effects of drugs in oral keratinocytes, as measured by western blot, IHC and quantitative PCR methods. We have road tested this methodology in our Phase I trial of perifosine (see below). In order to fully validate the use of oral keratinocytes obtained by cytobrush, we have opened in October 2002 a protocol in our institute -Molecular, Genetic, and Biochemical Effects Of Novel Therapies In Buccal Mucosal Cells -(03-D-0115) This protocol will test the pharmacodynamic effects of novel agents in oral buccal mucosal cells in patients already enrolled in Phase I and II clinical trials for neoplastic diseases at the Clinical Center. In this study we conduct, in parallel, cytobrushes and punch biopsies from buccal mucosa, at similar time points (before and after therapy) to compare the gene and protein expression profile of those tissues. Some of the subjects will also undergo tumor biopsy, if accessible or available. Genomic and proteomic profiles and post-translational protein modifications will be determined as well. DNA will be extracted from these tissues and will be screened for genetic polymorphisms in several candidate alleles that can affect drug action. Clinical effects (i.e. response, toxicity) will be correlated with biochemical/molecular profiles obtained from keratinocytes (brushes and punch biopsies), PBMCs, and tumors samples, if available. If successful, we will be in a position to export this technology to the extramural world to test and dose novel agents in a more rational way, hopefully, increasing their therapeutic indices. Based on our previous preclinical and clinical studies with flavopiridol, in collaboration with NCI/NIDCD, we are currently accruing patients to our protocol entitled -Phase II trial of daily bolus flavopiridol for five consecutive days in patients with recurrent/metastatic squamous cell carcinoma of the Head and Neck (SCCHN)-. Thus far, we have treated only three patients. We anticipate to complete the first phase of the study (total of 9 eligible patients) in the next 6 months. If a response is observed, we will expand the cohort to a total of 23 eligible patients (second phase). We have completed a Phase I trial of perifosine, in collaboration with NCI. Preliminary translational studies in patients receiving this agent demonstrated that, similar to what was observed in in vitro models in our unit, a clear induction of p21waf1 at the protein/mRNA level was observed in oral keratinocytes and lymphocytes obtained from these patients. As mentioned above, we are collaborating with NCI in a Phase 2 trial of perifosine in patients with metastatic prostate cancer, in order to validate these initial translational results. We are establishing a new NIDCR clinical/translational program for the diagnosis, prognosis and treatment of premalignant oral lesions (leukoplakia). Protocols involving small molecules for the treatment and prevention of leukoplakia are under review. In collaboration with Alter Blanche (NCI), we are participating in collaborative effort to study prospectively a large cohort of patients afflicted with Fanconi Anemia. Patients with Fanconi anemia who survive the hematological complications succumb, in the majority of cases, due to HNSCC. We are evaluating these patients to prospectively determine the molecular and biochemical factors responsible for the malignant transformation in the oropharyngeal tissues.
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