Changes in the concentration of EGFR-mutated plasma DNA in the first hours of targeted therapy allow the prediction of tumor response in patients with EGFR-driven lung cancer.

PURPOSE: This study aimed to analyze changes in the plasma concentration of EGFR-mutated circulating tumor DNA (ctDNA) occurring immediately after the start of therapy with EGFR tyrosine kinase inhibitors (TKIs). METHODS: Serial plasma samples were collected from 30 patients with EGFR-driven non-small cell lung cancer before intake of the first tablet and at 0.5, 1, 2, 3, 6, 12, 24, 36 and 48 h after the start of the therapy. The content of EGFR alleles (exon 19 deletions or L858R) in ctDNA was measured by ddPCR. RESULTS: ctDNA was detected at base-line in 25/30 (83%) subjects. Twelve (50%) out of 24 informative patients showed > 25% reduction of the ctDNA content at 48 h time point; all these patients demonstrated disease control after 4 and 8-12 weeks of therapy. The remaining 12 individuals showed either stable content of EGFR-mutated ctDNA (n = 5) or the elevation of ctDNA concentration (n = 7). 10 of 12 patients with elevated or stable ctDNA level achieved an objective response at 4 weeks, but only 5 of 10 evaluable patients still demonstrated disease control at 8-12 weeks (p = 0.032, when compared to the group with ctDNA decrease). The decline of the amount of circulating EGFR mutant copies at 48 h also correlated with longer progression-free survival (14.7 months vs. 8.5 months, p = 0.013). CONCLUSION: Comparison of concentration of EGFR-mutated ctDNA at base-line and at 48 h after the start of therapy is predictive for the duration of TKI efficacy.

Content of circulating tumor DNA depends on the tumor type and the dynamics of tumor size, but is not influenced significantly by physical exercise, time of the day or recent meal.

PURPOSE: This study aimed to investigate factors, which influence the content of circulating tumor DNA (ctDNA). METHODS: 398 serial plasma samples were collected within 1-7 consecutive days from patients with EGFR-mutated lung cancer (n = 13), RAS/RAF-mutated colorectal cancer (n = 54) and BRAF-mutated melanoma (n = 17), who presented with measurable tumor disease. The amount of ctDNA was determined by ddPCR. RESULTS: Among 82 patients, who donated 2-6 serial plasma samples, 42 subjects were classified as ctDNA-positive; only 22% cases were mutation-positive across all consecutive tests, while 24/82 (29%) patients showed presence of mutated ctDNA in some but not all blood draws. Subjects with progressing tumors had higher probability of being detected ctDNA-positive as compared to patients, who responded to therapy or had stable disease (39/55 (71%) vs. 4/24 (17%); p = 0.0001). Our study failed to reveal the impact of the time of the day, recent meal or prior physical exercise on the results of ctDNA testing. CONCLUSIONS: Presence of ctDNA in plasma is particularly characteristic for patients, who experience clinical progression of tumor disease. Consecutive plasma tests may occasionally provide discordant data; thus, the repetition of analysis may be advised in certain cases in order to ensure the validity of negative ctDNA result.

Comparative analysis of expression of mutant and wild-type alleles is essential for reliable PCR-based detection of MET exon 14 skipping.

MET exon 14 skipping (exon 14Δ) mutations are associated with tumor sensitivity to a number of tyrosine kinase inhibitors, however clinical testing for MET gene status remains complicated. We developed a simple allele-specific PCR cDNA-based test, which allowed for the identification of MET exon 14Δ allele in 35 (2.5%) out of 1415 EGFR mutation-negative lung carcinomas (LCs). MET exon 14Δ was significantly associated with elderly age and non-smoking status of the patients. A total of 34 (97%) out of 35 tumors carrying MET exon 14Δ showed preferential expression of the mutated allele; this imbalance was attributed to the down-regulation of the expression of the wild-type gene copy. Sanger sequencing confirmed the presence of genomic exon 14 splice site mutations in 24/35 (68.6%) cases, which showed MET exon 14 skipping by PCR. In addition to LCs described above, some carcinomas demonstrated low-abundance MET exon 14Δ-specific signal. Low-level expression of MET exon 14Δ allele may potentially compromise the results of allele-specific PCR-based tests, therefore comparison of the level of expression of mutated and normal alleles is essential for the reliability of MET gene testing.

Variability in lung cancer response to ALK inhibitors cannot be explained by the diversity of ALK fusion variants.

Multiple laboratory evidences indicate that distinct variants of ALK translocations differ in their biochemical properties and responsiveness to ALK tyrosine kinase inhibitors (TKIs). These data are supported by some clinical studies, which showed improved responses to crizotinib in non-small cell lung cancer (NSCLC) patients carrying particular variants of ALK translocation. We retrospectively considered 64 Russian patients with ALK-rearranged NSCLC, who were treated by crizotinib (n = 23), ceritinib (n = 39) or alectinib (n = 2). ALK fusion variants were genotyped by PCR. Median progression-free survival (PFS) approached to 18 and 21 months in subjects with "short" (v.3a/b, v.5a/b) vs. "long" (TAPE-domain containing) fusion variants (p = 0.783), respectively; similar data were obtained while comparing EML4/ALK variant 1 vs. other ALK translocations (19 and 21 months, respectively; p = 0.604). Objective response rates were also strikingly similar in the above groups ("short": 88%, "long": 77%, p = 0.479; variant 1: 76%, other translocations: 81%, p = 0.753). Furthermore, ALK variants did not influence the disease outcomes when patients treated by crizotinib and ceritinib were analyzed separately. Overall, PFS on ALK TKI did not depend on whether the drug was administered upfront or after chemotherapy. Ceritinib produced significantly longer PFS than crizotinib (p = 0.022). In conclusion, this study revealed that distinct ALK translocation variants render similar clinical responsiveness to ALK inhibitors.