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Tesi etd-09182019-152705

Thesis type
Tesi di dottorato di ricerca
Application of liquid biopsy to the monitoring of response to targeted therapy and immunotherapy in cancer patients.
Settore scientifico disciplinare
Corso di studi
tutor Prof. Danesi, Romano
Parole chiave
  • glioma
  • exosomes
  • treatment monitoring
  • drug resistance
  • liquid biopsy
  • circulating free DNA
Data inizio appello
Data di rilascio
Riassunto analitico
In oncology to realize the so-called “precision medicine” the greatest challenge is to identify clinically significant variants that could be implemented in clinical practice “to provide the right dose of the right drug for the right patient at the right time”. Thus, the critical step is the characterization of the genetic profile of both cancer and individual, to decide which therapies a particular patient may benefit from, to discontinue the ineffective treatment, and to switch to another better therapy. Cell-free DNA (cfDNA) and exosomes-derived RNA can be extracted from the cell-free fraction of the blood, and provide a snapshot of the genetic profiles of primary and metastatic tumour sites, also called “liquid biopsy”. Although the detection of targetable mutations in cancer patients can be performed on tumour tissue, the monitoring of predictive biomarkers over the time to guide treatment selection is crucial, and liquid biopsy is a viable option for clinical practice.
This work means greater reliance on the application of liquid biopsy in the diagnosis of resistance to targeted therapy and immunotherapy in Non-Small Cell Lung Cancer (NSCLC) patients and in the characterization of the genetic profile of brain tumours.
In this study, 216 EGFR- and 20 ALK-mutated advanced NSCLC patients at progression to first-line treatment with EGFR- or ALK- tyrosine kinase inhibitors (TKIs), and 65 patients affected by brain tumours (64 grade II-IV gliomas and 1 ganglioglioma) were enrolled. Cell free circulating DNA (cfDNA) and exosome-derived RNA were extracted from plasma samples and the quantitative analysis of predictive and prognostic circulating biomarkers (i.e. point mutations in EGFR and ALK genes and PD-L1 mRNA in NSCLC patients or IDH1 and BRAF in brain tumours) was performed by digital droplet PCR.
In NSCLC patients the third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs) osimertinib was effective in patients positive for p.T790M mutation despite at the time of progression to the first line EGFR-TKI treatment the amount in plasma of this resistance mutation resulted much lower than the activating EGFR mutations (p<0.0001). The activating EGFR mutated allele fraction (act-EGFR MAF) and p.T790M/act-EGFR MAF ratio were significantly correlated with disease response (p = 0.02) and PFS [10 months vs not reached for patients with the act-EGFR MAF > 2.6% and < 2.6%, respectively (p=0.03) and 6 months vs. not reached for patients with the T790M/act-EGFR ratio ≤ 0.22 and > 0.22, respectively (p = 0.01)]. ALK secondary mutations were identified on cfDNA extracted from plasma of NSCLC patients at progression of disease to crizotinib. cfDNA was monitored after the switch of the ineffective therapy to the second-generation ALK inhibitors and the amount of ALK as well as KRAS mutations decreased along with tumour regression. Despite the tumour re-biopsy after progression to EGFR- or ALK TKIs remains a crucial issue for understanding tumour biology and histologic transformation, our study confirms the importance to test in plasma the act-EGFR MAF, T790M/act-EGFR MAF ratio and ALK resistance point mutations as surrogate biomarkers for prognosis and response prediction in NSCLC setting. Interestingly, this study demonstrates that dynamic measurement of PD-L1 expression in plasma-derived exosomes is feasible and may provide useful information on the response to treatment with anti-PD-1 antibodies. Finally, despite the case report described in this study demonstrated that exosomes may be a better vehicle of mutated allele in cerebral tumours compared to cfDNA, allowing the prediction of tumour progression and resistance to treatment, additional prospective studies are needed to evaluate whether circulating biomarkers, integrated with sequential imaging analysis, may improve the management of patients with brain tumour.