The Application of Real-Time PCR Technique to Detect Rare Cell Clones with Primary T790M Substitution of EGFR Gene in Metastases of Non-small Cell Lung Cancer to Central Nervous System in Chemotherapy Naive Patients

Abstract

The time-limited efficacy of reversible EGFR-TKIs in patients with advanced non-small cell lung cancer (NSCLC) with EGFR gene activating mutations is associated with development of treatment resistance after some period of therapy. This resistance predominantly results from secondary mutations located in EGFR gene, especially T790M substitution. There is limited information available concerning the prevalence of primary T790M mutations in patients with metastatic NSCLC tumors before treatment with EGFR-TKIs. The aim of work was to assess the prevalence of de novo T790M mutations in EGFR gene in tissue samples from NSCLC metastatases in central nervous system (CNS) in both chemotherapy and EGFR-TKI naive NSCLC patients. We analyzed DNA samples isolated from paraffin-embedded tissue from CNS metastases for T790M mutations using real-time PCR and TaqMan probe against the T790M mutant sequence. The tissue samples were taken during palliative neurosurgery in 143 NSCLC patients. Amplification of the T790M-specific sequence was detected in 25 patients (17.5 %). The quantity of mutated DNA was less than 1 % in all samples with amplification, and in vast majority (20 patients, 14 % of all samples) it was even less that 0.1 %. In 5 patients (3.5 %) quantity of mutated DNA ranged from 0.1 to 1 % and true positive results of T790M mutation presence in these patients were most possible. Amplification of this sequence was not concurrent with common EGFR mutations and was not associated with sex, smoking status and pathological type of cancer. There is a possibility to detect the primary T790M mutation in brain metastases of NSCLC in EGFR-TKIs naïve patients.

Novel approaches against epidermal growth factor receptor tyrosine kinase inhibitor resistance

“…Baseline T790M mutations have been detected in EGFR TKI-naïve patients, and in brain metastases, at low rates (<1%) using standard molecular analysis [ 31 , 132 ]. Data obtained using more sensitive methodology (e.g.…”

“…Data obtained using more sensitive methodology (e.g. MALDI-TOF mass spectrometry, TaqMan quantitative PCR, amplification refractory mutation system) suggest that the prevalence of de novo mutations may be much higher (22–25%) [ 132 , 133 ], although replication in larger patient samples is warranted before firm conclusions can be drawn. Regardless of prevalence, given that approved, first-line EGFR TKI therapies have proven to be of limited value in these patients [ 31 , 134 ], the possibility of such mutations should be considered in any diagnostic approach.…”

Aktualizacja danych dotyczących skuteczności odwracalnych inhibitorów kinazy tyrozynowej EGFR u chorych na NDRP z mutacjami aktywującymi genu EGFR z uwzględnieniem częstych i rzadkich mutacji

“…Oh i wsp., metodą PNA PCR Clamp, wykryli mutację T790M u 8,2% chorych na NDRP, Maheswaran i wsp., metodą SARMS (scorpion amplifi cation refractory mutation system) real-time PCR -u 38% chorych nieleczonych IKT EGFR, Inukai i wsp., metodą ME-PCR (mutant enriched PCR) -u 3,2% pacjentów, a Su i wsp., metodą MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-fl ight mass spectrometry) -u 27,8% chorych nieleczonych IKT EGFR [31][32][33][34]. Nasze badanie, z wykorzystaniem specyfi cznych sond molekularnych i techniki real-time PCR, pozwoliło na wykrycie pierwotnej mutacji T790M u 3,5% polskich chorych na NDRP [35].…”