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Chemoresistance profile of circulating tumor cells: Toward a clinical benefit?

2008; Wiley; Volume: 123; Issue: 7 Linguagem: Inglês

10.1002/ijc.23699

1097-0215

Paola Gazzaniga, Angela Gradilone, Giuseppe Naso, Enrico Cortesi, Walter Gianni, Luigi Frati, Anna Maria Aglianò,

Metastasis and carcinoma case studies

The detection, characterization and count of circulating tumor cells (CTC) in cancer patients, although intriguing and promising, still raises some opened questions.1 After the pioneer investigation performed by Cristofanilli,2 showing that the number of CTC before treatment is an independent predictor of progression-free survival and overall survival in patients with metastatic breast cancer, further investigations have demonstrated that CTC have superior and independent prognostic value of tumor burden3 and better correlate with overall survival than do changes determined by traditional radiology.4 All these new concepts open the era of the “microscopic” revolution, a definition used by Cristofanilli in 2005.5 Despite the undoubted prognostic significance of CTC count, oncologists are still wondering on the real significance of CTC, focusing their attention on a crucial point, the clinical benefit.6 Although several clinical trials have recently demonstrated the correlation between CTC count and prognosis of patients,7, 8 some questions are still unsolved, such as the nature of these cells and, more important, the therapeutic consequences of their detection and enumeration. Specifically, the question of what to do when CTC levels rise in course of treatment still remains unanswered. The hypothetical solutions could be the switch to a different regimen, other than the completion of the planned course, or the addition of new agents. In this context, we feel to add a new question: “switch to different regimens, according to which rules”? To address this last question, we believe it would be important, besides the count of CTC, the characterization of their drug-resistance profile. For this purpose, in January 2008, we started a study (actually ongoing) with the aim to investigate the chemoresistance profile of CTC isolated by peripheral blood of cancer patients. Briefly, peripheral blood samples are obtained with informed consent from all patients with solid tumors attending the Division of Medical Oncology who accept to participate to the study. From January to date, 30 patients with stage IV cancer scheduled to receive standard systemic therapy have been enrolled (7 bladder cancer, 13 breast cancer, 5 colon cancer, 5 NSCLC). CTCs are isolated from 10 cc of peripheral blood by CELLection™ Dynabeads® coated with the monoclonal antibody towards the human Epithelial Cell Adhesion Molecule (EpCam); RNA is then extracted by Trizol (Invitrogen, Carlsbad, CA) and subjected to reverse transcription assay. The evaluate the suitability of the extracted RNAs, RT-PCR assay with GAPDH primers as internal control are performed, while the presence of epithelial cells in the pellet obtained is routinely confirmed using primers specific for epidermal growth factor receptor (EGFR). Each sample found positive for CTC presence is further evaluated for the expression of a panel of ATP-binding-cassette transporters involved in the resistance to standard chemotherapic drugs; specifically multidrug resistance protein 2 (MRP2), which confers resistance to doxorubicin, epirubicin, etoposide, vinca alkaloids; MRP4 (resistance to irinotecan, topotecan, methotrexate); MRP5 (resistance to 5-fluorouracil, cisplatin); MRP7 (resistance to taxanes9). Resistance to gemcitabine is investigated through the analysis of hENT1 × dCK/RRM1 × RRM2 expression.10 According to this type of CTC analysis, each patient, even with the same tumor type and staging, is characterized by his own, specific chemoresistance profile, which allow us to trace a hypothetical individual optimal regimen to be adopted in case of failure of the standard treatment. In Figure 1, the chemoresistance profile obtained on CTC of three exemplificative patients is shown. RT-PCR amplification products obtained by CTC isolated by three cancer patients loaded on 2% agarose gel. + : positive control (M14 cell line for EGFR, MRP2, MRP5,MRP7; Colo-699 cell line for MRP4; 5637 cell line for dCK, hENT and RR); M: molecular marker. On the right, the panel of drug resistance induced by each MRP. During the planned follow-up, we will evaluate for each patient the response to standard regimens by RECIST criteria, and we will try to correlate it with the molecular chemoresistance profile on the basis of CTC characterization. This approach could represent the first step towards a desirable trial that includes a change in therapy based on the CTC results, where patients would be randomized to continue therapy versus stopping the initial drugs and changing agents. Again, it is to be hoped that any hypothetical change in therapy on the basis of CTC chemoresistance profile, should be strictly individualized in order to guide the oncologist in the right choice, avoiding unuseful and toxic regimens. Yours sincerely, Paola Gazzaniga, Angela Gradilone, Giuseppe Naso, Enrico Cortesi, Walter Gianni, Luigi Frati, Anna Maria Aglianò.