Effect of Osimertinib on CTCs and ctDNA in EGFR Mutant Non-Small Cell Lung Cancer Patients: The Prognostic Relevance of Liquid Biopsy.

INTRODUCTION: Liquid biopsy is a useful tool for monitoring treatment outcome in solid tumors, including lung cancer. The relevance of monitoring CTCs and plasma ctDNA as predictors of clinical outcome was assessed in EGFR-mutant NSCLC patients treated with osimertinib. METHODS: Forty-seven EGFR-mutant NSCLC patients who had progressed on prior first- or second-generation EGFR inhibitors were enrolled in the study and treated with osimertinib, irrespective of the presence of the T790M mutation in the primary tumor or the plasma. Peripheral blood was collected at baseline (n = 47), post-Cycle 1 (n = 47), and at the end of treatment (EOT; n = 39). CTCs were evaluated in 32 patients at the same time points (n = 32, n = 27, and n = 21, respectively) and phenotypic characterization was performed using triple immunofluorescence staining (CK/VIM/CD45). RESULTS: Osimertinib resulted in an ORR of 34% (2 CR) and a DCR of 76.6%. The median PFS and OS values were 7.5 (range, 0.8-52.8) and 15.1 (range, 2.1-52.8) months, respectively. ctDNA was detected in 61.7%, 27.7%, and 61.5% of patients at baseline, post-Cycle 1, and EOT, respectively. CTCs (CK+/CD45-) were detected in 68.8%, 48.1%, and 61.9% of patients at the three time points, respectively. CTCs expressing both epithelial and mesenchymal markers (CK+/VIM+/CD45-) were detected in 56.3% and 29.6% of patients at baseline and post-Cycle 1, respectively. The detection of ctDNA at baseline and post-Cycle 1 was associated with shorter PFS and OS, whereas the ctDNA clearance post-Cycle 1 resulted in a significantly longer PFS and OS. Multivariate analysis revealed that male sex and the detection of ctDNA at baseline were independent predictors of shorter PFS (HR: 2.6, 95% C.I.: 1.2-5.5, p = 0.015 and HR: 3.0, 95% C.I.: 1.3-6.9; p = 0.009, respectively). CONCLUSIONS: The decrease in both CTCs and ctDNA occurring early during osimertinib treatment is predictive of better outcome, implying that liquid biopsy monitoring may be a valuable tool for the assessment of treatment efficacy.

KMT2C promoter methylation in plasma-circulating tumor DNA is a prognostic biomarker in non-small cell lung cancer.

MLL3 histone methyltransferase, encoded by the KMT2C gene, is a tumor suppressor that has an essential role in cell-type-specific gene expression. We evaluated the prognostic significance of KMT2C promoter methylation as a circulating epigenetic biomarker in plasma cell-free DNA (cfDNA) in non-small cell lung cancer (NSCLC). We examined the methylation status of KMT2C promoter using a novel highly specific and sensitive real-time methylation-specific PCR (MSP) assay in (a) operable NSCLC: 48 fresh-frozen NSCLC tissues, their corresponding adjacent non-neoplastic tissues, and 48 matched plasma samples; (b) metastatic NSCLC: 91 plasma samples; and (c) 60 plasma samples from healthy donors (HD). KMT2C promoter methylation in plasma cfDNA was detected in 7/48 (14.6%) patients with operable and in 18/91 (19.8%) patients with advanced NSCLC but in none (0/60, 0%) of the plasma samples from HD. In operable NSCLC, in corresponding adjacent non-neoplastic tissue samples, KMT2C promoter methylation was detected in 3/48 (6.3%) cases. Moreover, in operable NSCLC, KMT2C promoter methylation in plasma cfDNA was related to reduced disease-free survival (ΗR = 0.239; P = 0.001) and worse overall survival (OS; HR = 0.342, P = 0.023). In metastatic NSCLC, KMT2C promoter methylation in plasma cfDNA was related to worse progression-free survival (PFS; HR = 0.431; P = 0.005) and worse OS (HR = 0.306; P < 0.001). Our data strongly suggest that the detection of KMT2C promoter methylation in plasma cfDNA predicts poor prognosis in patients with both operable and metastatic NSCLCs. KMT2C promoter methylation in plasma cfDNA therefore merits further evaluation and validation as a noninvasive circulating epigenetic biomarker.

SOX17 promoter methylation in plasma circulating tumor DNA of patients with non-small cell lung cancer.

BACKGROUND: SOX17 belongs to the high-mobility group-box transcription factor superfamily and down-regulates the Wnt pathway. The aim of our study was to evaluate the prognostic significance of SOX17 promoter methylation in circulating tumor DNA (ctDNA) in plasma of non-small cell lung cancer (NSCLC) patients. METHODS: We examined the methylation status of SOX17 promoter in 57 operable NSCLC primary tumors and paired adjacent non-cancerous tissues and in ctDNA isolated from 48 corresponding plasma samples as well as in plasma from 74 patients with advanced NSCLC and 49 healthy individuals. SOX17 promoter methylation was examined by Methylation Specific PCR (MSP). RESULTS: In operable NSCLC, SOX17 promoter was fully methylated in primary tumors (57/57, 100%), and in corresponding ctDNA (27/48, 56.2%) while it was detected in only 1/49 (2.0%) healthy individuals. In advanced NSCLC, SOX17 promoter was methylated in ctDNA in 27/74 (36.4%) patients and OS was significantly different in favor of patients with non-methylated SOX17 promoter (p=0.012). Multivariate analysis revealed that SOX17 promoter methylation in ctDNA was an independent prognostic factor associated with OS in patients with advanced but not operable NSCLC. CONCLUSIONS: Our results show that SOX17 promoter is highly methylated in primary tumors and in corresponding plasma samples both in operable and advanced NSCLC. In the advanced setting, SOX17 promoter methylation in plasma ctDNA has a statistical significant influence on NSCLC patient's survival time. Detection of SOX17 promoter methylation in plasma provides prognostic information and merits to be further evaluated as a circulating tumor biomarker in patients with operable and advanced NSCLC.

Phase III study comparing sequential versus alternate administration of cisplatin-etoposide and topotecan as first-line treatment in small cell lung cancer.

AIM: To compare the efficacy and tolerance of sequential versus alternate front-line administration of cisplatin-etoposide (PE) and topotecan (T) in patients with extensive stage small cell lung cancer (SCLC). PATIENTS AND METHODS: Patients were randomized to receive either 4 cycles PE (cisplatin 80 mg/m(2) i.v. day 1 and etoposide100 mg/m(2)/d i.v. days 1-3 every 21 days) followed by 4 cycles T (1.5 mg/m(2)/d i.v. days 1-5 every 21 days) (arm A, 183 patients) or the same regimens using an alternate sequence (arm B, 181 patients). RESULTS: There was no significant difference in terms of compliance with treatment, overall response rates (51.4% vs. 55.2%; p=0.458), median response duration (4.3 vs. 5.2 months; p=0.780), median time to tumour progression (5.7 vs. 6.4 months; p=0.494), median overall survival (10.9 vs. 9.8 months; p=0.186) and 1-year survival (43.8% vs. 36.5%) between the two arms. The incidence of severe grade 3-4 haematological and grade 2-4 non-haematological (asthenia, mucositis, diarrhoea and neurotoxicity) toxicity was comparable between the two arms. CONCLUSION: The comparison of sequential versus alternate administration of cisplatin-etoposide and topotecan as front-line treatment of patients with extensive stage SCLC revealed no clinically meaningful differences in terms of efficacy and tolerance.

Prognostic value of mature microRNA-21 and microRNA-205 overexpression in non-small cell lung cancer by quantitative real-time RT-PCR.

BACKGROUND: microRNA (miRNA) expression profiles are being intensively investigated for their involvement in carcinogenesis. We evaluated the prognostic value of mature microRNA-21 (miR-21) and mature microRNA-205 (miR-205) overexpression in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: We studied 48 pairs of NSCLC fresh frozen tissue specimens collected at time of surgery and before chemotherapy. Highly specific amplification and quantification of mature miR-21 and mature miR-205 was achieved using looped real time RT-PCR. RESULTS: miRNA expression, determined by real time RT-PCR, was defined by DeltaDeltaCt measurements. We detected overexpression of mature miR-21 in 25 (52.0%) of the 48 NSCLC paired specimens and overexpression of miR-205 in 31 (64.6%). Overexpression was assessed after comparison of miRNA expression in NSCLC tissues and in their corresponding noncancerous tissues with respect to U6 expression. During the follow-up period, 29 of 48 (60.4%) patients relapsed, and 23 of 48 died (47.9%). Mature miR-21 was upregulated in 16 of 29 (55.2%) patients who relapsed and 15 of 23 (65.2%) patients who died. Mature miR-205 was overexpressed in 19 of 29 patients who relapsed (65.5%) and 15 of 23 patients who died (65.2%). Mature miR-21 overexpression correlated with overall survival (OS) of the patients (P = 0.027), whereas overexpression of mature miR-205 did not. CONCLUSIONS: Our results suggest that overexpression of mature miR-21 is an independent negative prognostic factor for OS in NSCLC patients.

Quantitative real-time reverse transcription PCR study of the expression of vascular endothelial growth factor (VEGF) splice variants and VEGF receptors (VEGFR-1 and VEGFR-2) in non small cell lung cancer.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and its expression is increased in non-small cell lung cancer (NSCLC). We aimed to determine the expression pattern of VEGF splice variants in NSCLC and its correlation with the clinicopathological characteristics of tumors. METHODS: We used real-time reverse transcription PCR to quantify the mRNA expression of total VEGF, 4 VEGF splice variants (VEGF(121), VEGF(165), VEGF(183), and VEGF(189)), and 2 VEGF receptors (VEGFR-1 and VEGFR-2) in 27 pairs of cancerous and adjacent noncancerous tissues originating from patients with NSCLC. RESULTS: Total VEGF, VEGF(121), and VEGF(165) were expressed in all specimens, whereas VEGF(183) and VEGF(189) were present in small amounts in certain samples. Total VEGF, VEGF(121), and VEGF(165) mRNA was upregulated in cancerous compared with healthy tissues, whereas VEGF(183) and VEGF(189) expression tended to be higher in healthy tissues. The expression of VEGFRs was similar between matched specimens. No correlation was found between the expression of total VEGF or VEGF splice variants and the clinicopathological characteristics of tumors. The expression patterns of VEGF splice variants differed between tissue pairs. VEGF(121) was the major variant expressed in all samples; however, its relative expression was higher in cancerous tissues. The relative expression of VEGF(183) and VEGF(189) was upregulated in healthy lung tissues, whereas the ratio of VEGF(165) to total VEGF was similar between matched specimens. CONCLUSIONS: The expression pattern of certain VEGF splice variants is altered during tumorigenesis. Our data support the hypothesis that during malignant progression an angiogenic switch favoring the shorter diffusible isoforms occurs.

Real-time RT-PCR quantification of human telomerase reverse transcriptase splice variants in tumor cell lines and non-small cell lung cancer.

BACKGROUND: We developed and validated a real-time reverse transcription (RT)-PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants (alpha+beta+, alpha-beta+, alpha+beta-, alpha-beta-) in tumor cell lines and non-small cell lung cancer (NSCLC). METHODS: We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript-specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients. RESULTS: In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The alpha+beta- splice variant showed the highest expression and alpha-beta+ and alpha-beta- the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the alpha+beta- variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by alpha+beta+, with the alpha-beta+ and alpha-beta- splice variants having the lowest expression. In the NSCLC tumors, the alpha+beta+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival. CONCLUSIONS: Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT alpha+beta+ splice variant may be an independent negative prognostic factor for NSCLC patients.