New Methodological Approaches in Assessing and Monitoring the Targeted Treatment of Chordoma
The Liddy Shriver Sarcoma Initiative is pleased to announce the funding of a research project being undertaken by Dr. Silvana Pilotti in the Laboratory of Experimental Molecular Pathology of the Department of Pathology at the Fondazione IRCCS Istituto dei Tumori in Milan, Italy. The $25,000 grant is being made through the generosity of Stephen, Sally, and Eric Norcross in loving memory of Alison Norcross and through the generosity of Katherine Stadler and the former Bone Cancer International organization. The research study, "New methodological approaches in assessing and monitoring the targeted treatment of chordoma," will begin shortly.
Chordoma is a very rare sarcoma. Dr. Pilotti told us that chordomas have an incidence estimated around 1/1,000,000. She went on to say:
They originate from remnants of the fetal notochord and mainly affect the sacrum, skull base region and, in a minority of cases, the cervical and thoraco-lumbar vertebrae. Their localization makes the treatment of primary disease highly challenging in most cases. Surgery, the standard treatment, is often not effective in curing the disease. For this reason local relapse is common and affects the prognosis of most chordoma patients in the long run.
Pilotti notes that classical cytogenetic studies define these tumors as complex sarcomas because they are generally characterized by complex karyotypes. One of the most frequently reported events is the deletion of p36.31-1p36.13 region of chromosome 1. In relation to cell-cycle controlling genes, an immunophenotype-based study has found an alteration in the G(1)S checkpoint, principally restricted to p53 overexpression. Very little is known about receptor tyrosine kinase (RTK) activation, but their expression could offer useful alternative therapeutic targets because it is well known that chordomas do not respond to radiotherapy or conventional chemotherapy.
Dr. Pilotti and her group demonstrated in a series of 31 chordomas that platelet-derived growth factor receptor, beta polypeptide (PDGFRB) is highly expressed and phosphorylated, whereas platelet-derived growth factor receptor, alpha polypeptide (PDGFRA) and KIT are less expressed but anyway phosphorylated and thus activated. These findings, together with the absence of gain of function mutations and the presence of the cognate ligands, strongly support the hypothesis that the activation mechanism working on these receptors is an autocrine/paracrine loop. No role seems to be played by gene amplification, but few cases were studied. In the light of Dr. Pilotti's group's findings, the clinical benefit observed in chordoma patients treated with imatinib seems to be due to the switching off of all three receptors, in a way recently defined as a "low oncogene addiction."
A phase II study is now ongoing at the Istituto dei Tumori in Milan and 50 chordoma patients are under imatinib treatment and their enrolment was based on the presence of an activated/phosphorylated PDGFRB. With this as background, the aims of the research study funded by this grant are to:
- Monitor, through an innovative methodology (flow cytometry applied to solid tumors), the imatinib pharmacological response in patients during the treatment.
- Verify, through short term cultures or chordoma cell lines, the efficacy of new RTK inhibitors alone or in combination with DNA damaging agents, in order to find a second line therapy for patients who develop a resistance to imatinib, a phenomenon already observed in some patients.
A brief expansion of these two aims follows.
In daily practice, a critical point for the medical oncologist is represented by the understanding if the administered drug is effectively working on the patient. For target therapy this point is represented by target switching off, i.e., the receptor tyrosine kinase becomes unphosphorylated (and thus not active) after the treatment. In maintaining the receptor into an unphosphorylated/inactive conformation relies the success of the therapy and this could be verified directly on small invasive fine needle aspirates (FNA) analysing the activation status of the receptor through a cytofluorimetric approach, a technology commonly used for haematopoietic disease but not yet explored for solid tumors. In fact, when compared with the traditional method for assessing protein phosphorylation (i.e. immunoprecipitation and Western blot), the ability to distinguish a subset in heterogeneous populations of cells (multiparametricity), represents the main advantage of flow cytometry. The use of flow phospo-specific antibodies combined with flow cytometry multiparametricity, might be an innovative tool for measuring the phosphorilation status of PDGFRB in chordoma fine needle aspirates taken at onset and during the target treatment. In order to set up the "phosflow cytometry" approach for measuring the degree of PDGFRB phosphorylations in chordoma samples, Dr. Pilotti and her group plan to optimize the experimental conditions using the short term cultures obtained from surgical samples, comparing the phosphoflow results to traditional immuoprecipitation/Western blot experiments.
The possibility to handle an in vitro model, representative of the tumor, will give Dr. Pilotti and her fellow researchers the opportunity to test new drugs and to verify i) the modulation of tumoral signal transduction pathways; ii) drug efficacy in terms of doses. Preliminary results, obtained in two patients showing progressive disease on imatinib treatment, showed an apparently re-established tumor response combining imatinib with cisplatinum. However, to achieve the best results with this drug combination, it is mandatory to find the treatment algorithm, considering on the one hand that chemotherapy targets only "cycling" cells, and on the other hand, that RTK switches off (PDGFRB, PDGFRA, KIT) induced by imatinib at the applied dose, generally leads to cell cycle block. To achieve this aim, there is the need to identify biomarkers (at preclinical level) or surrogated (via functional studies on skin biopsies) useful in the establishment of the biotherapic efficacious dose which differs from the maximum tolerated dose (MTD) of the classical chemotherapy. This could be achieved through the analysis of small, non-invasive FNA detecting the phosphorylation level of PDGFRB ("phosflow cytometry"/Western blotting also of the downstream effectors, AKT, PI3K, mTOR), all representing indirect measures of Imatinib inhibitory effect of the RTK. They intend to couple these parameters with the time window of cycling cells in scheduling the cisplatinum treatment.
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