Interestingly, clinical outcome was correlated with the number of ex19del ctDNA copies in plasma as it disappeared within a few weeks from the beginning of osimertinib and appeared again and continuously improved thereafter, anticipating tumor progression. ex lover19del disappeared from plasma but appeared again and continuously improved a few months later on anticipating tumor progression. Interestingly, the switch Octreotide in ex lover19del was much more pronounced than additional mutations, since T790M appeared 3?months after the increase of ex lover19del, and C797S was detectable a few weeks before clinical disease progression. Then the patient received cytotoxic chemotherapy, which was associated with a decrease in ex lover19del and disappearance of T790M and C797S; however, at disease progression, all EGFR mutations improved again in plasma together with MET amplification which was recognized by NGS. Conclusions The measurement of ex lover19del changes in ctDNA is definitely a simple and sensitive approach to monitor clinical end result to osimertinib and, potentially, to additional therapeutic interventions. strong class=”kwd-title” Keywords: Circulating tumor DNA, NSCLC, EGFR mutations, Treatment monitoring, EGFR-TKIs, Digital droplet PCR, NGS Background The presence of activating EGFR mutations, mainly ex19del, strongly predicts response to EGFR-TKIs; however, in 50C60% of these patients, resistance is acquired through the development of T790M, a second missense mutation of EGFR, which is indeed targeted by osimertinib [1]. Some individuals retain EGFR oncogene habit actually after progression to osimertinib, as they may develop the C797S resistance mutation [2, 3]. The analysis of circulating tumor DNA (ctDNA) is definitely a valuable approach to monitor the clonal development of tumors during treatment and to detect mutations capable of inducing resistance to EGFR-TKIs [4]. Actually if the analysis of tumor cells is required to select the appropriate treatment, it is indeed associated with several limitations, including invasiveness, failure to comprehensively capture tumor heterogeneity, and cells availability for mutational screening. For these reasons, the analysis of EGFR mutations in ctDNA has recently emerged as a reliable, noninvasive alternative approach, showing high concordance with cells molecular profile, with good level of sensitivity ( ?65%) and high specificity ( ?88%) [5]. Here, we statement a case of ctDNA monitoring during osimertinib treatment and after disease progression, which provides evidence of the reliability of time-dependent changes in?EGFR activating mutation to predict response to treatment. Case demonstration In October 2012, a 46-year-old female was referred to our center for the presence of a large mass (50??70?mm) in the first-class lobe of the remaining lung with homolateral pleural effusion. The patient was never smoker, without family history of malignancy and without comorbidity. The cytological Octreotide analysis was made using a CT-guided good needle aspiration of the primary tumor and exposed an adenocarcinoma of the lung (TTF1+, CK7+) with the EGFR ex19del mutation. A PET-CT shown the presence of liver and bone metastases and a nodule in the right breast, confirmed like a metastasis by good needle aspiration. The patient received zoledronic acid 4?mg every 28?days and gefitinib 250?mg daily since November 2012 obtaining a partial response (PR). In August 2013, a disease progression (PD) was recorded, with an increase in size of the primary tumor and size and quantity of liver metastases. A mind MRI revealed the presence of two cortical nodules, which were treated with stereotactic radiotherapy. The patient was enrolled in the Win over trial and received 6?cycles of cisplatin and pemetrexed in addition gefitinib obtaining again a PR that lasted until June 2014. Thereafter, a new lung metastasis appeared in the superior lobe of the remaining lung and the mammary nodule improved in dimensions. From June 2014 to December 2014 the patient received erlotinib 150?mg daily obtaining an initial stabilization of the disease (SD); however, within 6?weeks, she experienced again a PD with the increase of the mammary nodule and the appearance of a new bone metastasis in the sacrum. In December 2014, EGFR ex lover19del and T790M mutations were detectable in a new needle biopsy of the primary tumor; only at this time a digital PCR-based method was available for the analysis of circulating tumor DNA (ctDNA). Briefly, the method was optimized in order to recover a suitable amount of ctDNA for molecular analysis from 3?ml of plasma using the QIAmp Circulating Nucleic Acid Kit (Qiagen?, Valencia, CA). ctDNA was examined using the.A written consent has been from the participant. Consent for publication A written consent has been from the participant for her clinical details to be published with this study. Competing interests The authors declare that they have no competing interests. Publishers Note Springer Nature remains neutral with regard to jurisdictional statements in published maps and institutional affiliations. Contributor Information Marzia Del Re, Email: moc.liamg@erled.aizram. Eleonora Rofi, Email: ti.dniwni@6010aronoele. Carla Cappelli, Email: moc.liamg@1illeppacalrac. Gianfranco Puppo, Email: ti.liamtoh@oppup.g. Stefania Crucitta, Email: moc.liamg@atticurc.ainafets. Simona Valeggi, Email: moc.liamg@47.01anomis. Antonio Chella, Email: ti.ilacsit@llek.otna. Romano Danesi, Telephone: +39050992632, Email: ti.ipinu@isenad.onamor. Iacopo Petrini, Email: ti.ipinu@inirtep.opocai.. and continuously improved a Octreotide few months later on anticipating tumor progression. Interestingly, the switch in ex lover19del was much more pronounced than additional mutations, since T790M appeared 3?months after the increase of ex lover19del, and C797S was detectable a few weeks before clinical disease progression. Then the patient received cytotoxic chemotherapy, which was associated with a decrease in ex lover19del and disappearance of T790M and C797S; however, at disease progression, all EGFR mutations improved again in plasma together with MET amplification which was recognized by NGS. Conclusions The measurement of ex lover19del changes in ctDNA is definitely a simple and sensitive approach to monitor clinical end result to osimertinib and, potentially, to additional therapeutic interventions. strong class=”kwd-title” Keywords: Circulating tumor DNA, NSCLC, EGFR mutations, Treatment monitoring, EGFR-TKIs, Digital droplet PCR, NGS Background The presence of activating EGFR Rabbit polyclonal to Neuropilin 1 mutations, primarily ex19del, strongly predicts response to EGFR-TKIs; however, in 50C60% of these patients, resistance is acquired through the development of T790M, a second missense mutation of EGFR, which is indeed targeted by osimertinib [1]. Some patients retain EGFR oncogene dependency even after progression to osimertinib, as they may develop the C797S resistance mutation [2, 3]. The analysis of circulating tumor DNA (ctDNA) is usually a valuable approach to monitor the clonal development of tumors during treatment and to detect mutations capable of inducing resistance to EGFR-TKIs [4]. Even if the analysis of tumor tissue is required to select the appropriate treatment, Octreotide it is indeed associated with several limitations, including invasiveness, failure to comprehensively capture tumor heterogeneity, and tissue availability for mutational screening. For these reasons, the analysis of EGFR mutations in ctDNA has recently emerged as a reliable, noninvasive alternative approach, showing high concordance with tissue molecular profile, with good sensitivity ( ?65%) and high specificity ( ?88%) [5]. Here, we report a case of ctDNA monitoring during osimertinib treatment and after disease progression, which provides evidence of the reliability of time-dependent changes in?EGFR activating mutation to predict response to treatment. Case presentation In October 2012, a 46-year-old woman was referred to our center for the presence of a large mass (50??70?mm) in the superior lobe of the left lung with homolateral pleural effusion. The patient was never smoker, without family history of malignancy and without comorbidity. The cytological diagnosis was made using a CT-guided fine needle aspiration of the primary tumor and revealed an adenocarcinoma of the lung (TTF1+, CK7+) with the EGFR ex19del mutation. A PET-CT exhibited the presence of liver and bone metastases and a nodule in the right breast, confirmed as a metastasis by fine needle aspiration. The patient received zoledronic acid 4?mg every 28?days and gefitinib 250?mg daily since November 2012 obtaining a partial response (PR). In August 2013, a disease progression (PD) was documented, with an increase in size of the primary tumor and size and quantity of liver metastases. A brain MRI revealed the presence of two cortical nodules, which were treated with stereotactic radiotherapy. The patient was enrolled in the IMPRESS trial and received 6?cycles of cisplatin and pemetrexed plus gefitinib obtaining again a PR that lasted until June 2014. Thereafter, a new lung metastasis appeared in the superior lobe of the left lung and the mammary nodule increased in dimensions. From June 2014 to December 2014 the patient received erlotinib 150?mg daily obtaining an initial stabilization of the disease (SD); however, within 6?months, she experienced again a PD with the increase of the mammary nodule and the appearance of a new bone metastasis in the sacrum. In December 2014, EGFR ex lover19del and T790M mutations were detectable in a new needle biopsy of the primary tumor; only at this time a digital PCR-based method was available for the analysis of circulating tumor DNA (ctDNA). Briefly, the method was optimized in order to recover a suitable amount of ctDNA for molecular analysis from 3?ml of plasma using the QIAmp Circulating Nucleic Acid Kit (Qiagen?, Valencia, CA). ctDNA was examined using the Prime PCR Probe Assay on a QX100? Droplet Digital? PCR System (BioRad?, Hercules, CA) for EGFR mutations (ex lover19del, T790M, and?C797S) [6]. The ctDNA sample was considered as EGFR mutant when at least one droplet was above the fluorescence intensity threshold of 3000 and results were reported as copies/ml. The first plasma specimen was obtained in December 2014 and confirmed the presence of ex19del and T790M mutations?(480 and 260 copies/ml, respectively; Fig.?1). The patient was treated with atezolizumab from March to May 2015 and received stereotactic radiotherapy around the lung main tumor and on.
