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KRAS oncogene mutations (MUTKRAS) drive resistance to EGFR inhibition by providing alternative signaling as demonstrated in colo-rectal cancer. In non-small cell lung cancer (NSCLC), the efficacy of treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) depends on activating EGFR mutations (MUTEGFR). However, inhibition of EGFR may select resistant cells displaying alternative signaling, i.e., KRAS, or restoration of EGFR activity due to additional MUTEGFR, i.e., the c.2369C > T (p.T790MEGFR).
The aim of this study was to investigate the appearance of MUTKRAS during EGFR-TKI treatment and their contribution to drug resistance.
This study used cell-free circulating tumor DNA (cftDNA) to evaluate the appearance of codon 12 MUTKRAS and p.T790MEGFR mutations in 33 advanced NSCLC patients progressing after an EGFR-TKI.
p.T790MEGFR was detected in 11 (33.3%) patients, MUTKRAS at codon 12 in 3 (9.1%) while both p.T790MEGFR and MUTKRAS codon 12 were found in 13 (39.4%) patients. Six patients (18.2%) were KRAS wild-type (WTKRAS) and negative for p.T790MEGFR. In 8 subjects paired tumor re-biopsy/plasma samples were available; the percent concordance of tissue/plasma was 62.5% for p.T790MEGFR and 37.5% for MUTKRAS. The analysis of time to progression (TTP) and overall survival (OS) in WTKRAS vs. MUTKRAS were not statistically different, even if there was a better survival with WTKRAS vs. MUTKRAS, i.e., TTP 14.4 vs. 11.4 months (p = 0.97) and OS 40.2 vs. 35.0 months (p = 0.56), respectively.
MUTKRAS could be an additional mechanism of escape from EGFR-TKI inhibition and cftDNA is a feasible approach to monitor the molecular development of drug resistance.
Activating mutations of epidermal growth factor receptor (MUTEGFR) predict sensitivity to tyrosine kinase inhibitors (TKI) in non-small cell lung cancer (NSCLC). Despite a very high response rate (about 70%) to first-line treatment with the EGFR-TKIs (erlotinib, gefitinib or afatinib) in MUTEGFR NSCLC, tumors invariably progress after a median of 9–13 months from the beginning of treatment [1–3].
The understanding of the molecular basis of acquired resistance to TKI  and its application to treatment monitoring may improve treatment management by discontinuing ineffective treatments and directing towards most appropriate second line options before clinical progression may occur. Indeed, EGFR signaling is maintained in most cases that develop secondary resistance  suggesting that additional molecular mechanisms can bypass EGFR-TKI inhibition reactivating the signaling pathway. Several mechanisms of acquired resistance to EGFR-TKI have been described after progression, including c.2369C > T (p.T790M) EGFR gatekeeper mutation (p.T790MEGFR, ~50% of patients) , MET (5–15%)  or HER2 (12%)  amplifications, PIK3CA (4.1%)  or BRAF (1%)  mutations or transformation into small cell histology (3%) .
NSCLC heterogeneity can drive the therapeutic decisions ; therefore, tissue availability is increasingly recognized as a crucial issue. Unfortunately, the location of the tumor and the risk of complications are serious limitations to re-biopsies in NSCLC . Alternatively, the detection of somatic mutations in cell-free tumor DNA (cftDNA) released in plasma could be instrumental for a better understanding of the genetic modifications driven by the selective pressure of drug treatments .
Interestingly, approximately 15–25% of patients with NSCLC have KRAS mutations (MUTKRAS), resulting in constitutive activation of KRAS signaling pathways. MUTKRAS is a negative predictor of benefit to anti-EGFR antibodies in colo-rectal cancer, while it seems to be a negative predictor of response to EGFR-TKIs in EGFR wild type (WTEGFR) NSCLC patients . In a previous study on a large collection of NSCLC tissues from patients with acquired resistance to EGFR-TKI, NRAS or KRAS mutations were not demonstrated . Despite these negative results, we employed a sensitive ddPCR-based platform to investigate the presence of MUTKRAS alleles in plasma of patients resistant to EGFR-TKIs and we were able to demonstrate a potential role of MUTKRAS in acquired resistance to EGFR-TKI, besides the p.T790MEGFR. This finding reveals a potential new mechanism of resistance to EGFR-TKI and underscores the need of a periodic monitoring of somatic mutations of known oncogenes to deliver the best personalized treatment in a timely fashion.
The clinical characteristics of patients are reported in Table Table1.1. Of 33 patients, 20 (60.6%) were female and 13 (39.4%) male. Median age was 62 years (range 41 – 75); 32 patients were affected by a stage IV disease, while one was a stage IIIB NSCLC. The frequency of activating MUTEGFR was as follows: 20 patients (60.6%) showed ex19delEGFR, 10 patients (30.3%) p.L858REGFR, 2 patients (6.1%) p.L747PEGFR and 1 patient presented ex19insEGFR (3%). As expected, the majority of them (66.7%) was never-smokers, while 9 (27.2%) and 2 (6.1%) patients were former- and current-smokers, respectively. Twenty-seven (81.8%) subjects received gefitinib and 6 (18.2%) erlotinib; the treatment was administered as first-line in 23 (69.7%) (including 2 as maintenance), second-line in 6 (18.2%) and third or further lines in 4 patients (12.1%). The majority of them (66.7%) presented partial response to TKI treatment and only 1 patient showed complete response (Table (Table1).1). Stable and progressive diseases were observed in 4 (12.1%) and 6 subjects (18.2%), respectively. Patients who have progressed on EGFR-TKI treatment, all receiving gefitinib, presented the following molecular profile in their primary tumors: p.L747PEGFR and ex19delEGFR (n = 1 each) and p.L858REGFR (n = 4). Median time to progression (TTP) was 13.6 months (95% Confidence Interval, CI, range 8.0 – 19.2 months) and median overall survival (OS) was 40.2 months (95% CI range 25.8–54.7 months) for the overall population.
The description of patients with activating MUTEGFR in their primary tumors as well as the percentages of p.T790MEGFR and MUTKRAS alleles in cftDNA at the time of EGFR-TKI progression is reported in Table Table2.2. In 16 patients (48.5%), a codon 12 MUTKRAS was detected in cftDNA (Figure (Figure1).1). In addition to this, the p.T790MEGFR (c.2369C > T) second site mutation was present in the cftDNA of 24 patients (72.7%). Interestingly, 13 patients (39.4%) had both the MUTKRAS and p.T790MEGFR, while 3 (9.1%) and 11 (33.3%) subjects displayed only MUTKRAS or p.T790MEGFR, respectively (Figure (Figure1).1). Six subjects displayed no mutations.
The association between smoking habit and the occurrence of MUTKRAS in cftDNA was investigated. Regarding the 11 patients with smoking history, 2 (18.2%) presented MUTKRAS and 9 (81.8%) were wild-type (WTKRAS). On the contrary, of 22 non-smoking subjects, 14 (63.3%) were MUTKRAS and 8 (36.4%) WTKRAS. Fisher's extact test revealed that non-smoking habit and MUTKRAS were significantly associated (p = 0.026).
In 8 patients, paired re-biopsies and cftDNA were available. The 8 re-biopsies were performed in a different tumor site with respect to the initial diagnosis, the choice being dependent on several factors, i.e., anatomical accessibility, new or progressing lesions. The analysis of re-biopsies by standard methods and ddPCR demonstrated p.T790MEGFR in 4 (standard) vs. 2 (ddPCR) samples and MUTKRAS in none (standard) vs. 3 (ddPCR) specimens. p.T790MEGFR and MUTKRAS were detected in 7 and 5 cftDNA specimens, respectively.
The analysis of KRAS status by ddPCR in the biopsies at diagnosis revealed the presence of MUTKRAS in 2 patients, who were WTKRAS by standard method. Therefore, the percentage of patients developing MUTKRAS as a mechanism of acquired resistance is 42% (14 patients). In terms of TTP and OS, there was no difference between these patients and the others (p = 0.19 and p = 0.13, respectively).
The concordance between tissue of re-biopsies (standard methods) and plasma (ddPCR), calculated by combining positive and negative results, was 62.5% and 37.5% for p.T790MEGFR and MUTKRAS, respectively (Table (Table3).3). Moreover, three paired samples found positive for the p.T790MEGFR on re-biopsies (standard methods) and on cftDNA (ddPCR), were negative by ddPCR on primary tissue (Table (Table33).
The analysis of survival data stratified according to KRAS status in cftDNA, showed that TTP and OS were not statistically different, despite a trend towards a better survival in WTKRAS vs. MUTKRAS patients, i.e., TTP 14.4 vs. 11.4 months (p = 0.97) and OS 40.2 vs. 35.0 months (p = 0.56), respectively.
Finally, samples were analysed also for codons 13 (p.G13D) and 61 MUTKRAS and MUTNRAS, and V600EBRAF and all the samples were found to be wild-type. Unfortunately, due to sample restriction, cftDNA was insufficient to perform the analysis of PI3K/Akt mutations.
The present study demonstrates the presence of MUTKRAS in the cftDNA of a significant proportion of patients progressing after EGFR-TKI treatment. In addition to this, the present study provides evidence that sensitizing MUTEGFR and MUTKRAS can coexist after the selective pressure of EGFR-TKI treatment.
p.T790MEGFR determines acquired resistance by increasing the affinity of EGFR to ATP [16, 17]. p.T790MEGFR has been described in re-biopsies of 50–63% of tumors progressing under EGFR-TKI treatment [4, 11] and in the cftDNA at a frequency similar to our study [18–20]. Because drugs active on p.T790MEGFR, such as AZD9291 and rociletinib [21, 22], are under clinical study and will be available soon in the clinical practice, the identification of this molecular marker is of utmost clinical relevance.
In our study, p.T790MEGFR was more frequent in L858REGFR patients than in ex19delEGFR ones (80% vs. 67%); on the contrary, MUTKRAS in cftDNA was detected in 55% of patients with ex19delEGFR vs. 30% of patients with L858REGFR. To our knowledge, a mechanism of resistance depending on activating MUTEGFR has not been previously reported; however, this cohort is too small to draw any conclusion.
MUTEGFR and MUTKRAS are mutually exclusive in primary NSCLC and only anecdotal case reports described their coexistence [23, 24]. MUTKRAS occurs in approximately 20% of NSCLC cases at diagnosis, more frequently in Caucasian population, adenocarcinomas, males and current smokers [25, 26]. About 90% of KRAS mutations occur in exon 2 (codon 12 and 13), while exon 3 (codon 61) is less frequently involved [26, 27]; in never-smokers with lung adenocarcinoma, MUTKRAS is more frequently a transition (G to A) compared to transversion in current smokers . Colo-rectal cancer cells with MUTKRAS treated with anti-EGFR monoclonal antibodies are able to escape growth inhibition by several mechanisms, including MUTRAS . While the role of MUTKRAS in primary resistance to EGFR-TKIs in molecularly unselected NSCLC is quite well established [29, 30], its development and role in acquired resistance to EGFR-TKIs in MUTEGFR patients has not been explored in detail. In a previous work on a large collection of NSCLC tissues from patients with acquired EGFR-TKI resistance, MUTNRAS or MUTKRAS were not demonstrated . However, comparison with the present results is not possible because detailed information were not provided neither on the timing of sampling with respect of development of TKI resistance nor on the type of tissue analysed. Therefore, we addressed this issue and a sensitive ddPCR-based platform was employed to investigate the presence of MUTKRAS alleles besides the well-known p.T790MEGFR. Due to its high sensitivity, ddPCR is able to identify small amounts of MUTKRAS and many methodological issues need to be addressed prospectically, particularly the threshold level of both MUTKRAS and p.T790MEGFR to be considered clinically relevant. However, a mechanism of drug resistance does not necessarily reflect biologic aggressiveness and the lack of difference in OS between KRAS wild-type and mutated patients it is therefore not surprising. The numeric dimension of the cell clone bearing MUTKRAS should be taken into consideration as well. Nevertheless, despite the low proportion of smokers in our cohort, the high prevalence of MUTKRAS could support its role of as mechanism of acquired resistance.
Eight patients underwent re-biopsy after tumor progression during EGFR-TKI, allowing a comparison between tissue and cftDNA. The detection of mutations in cftDNA but not in re-biopsy, using both standard methods and ddPCR, could suggest the presence of heterogeneity within metastatic sites or the lower performance of ddPCR in the presence of paraffin. Nevertheless, the detection of mutations in both plasma and tissue by ddPCR, but not by standard methods, could be due to the higher sensitivity of ddPCR analysis. Two patients, initially diagnosed WTKRAS by standard method, were re-analysed by ddPCR and were found MUTKRAS in the primary biopsy, suggesting that the MUTKRAS clone co-existed with activating MUTEGFR since the beginning, as also demonstrated in previous reports [23, 24]. In these patients, MUTKRAS cannot strictly be considered a mechanisms of resistance but it could be possible that EGFR-TKI treatment may have favored the expansion of MUTKRAS-positive clones. However, conclusions cannot be drawn as pre-treatment cftDNA was not available.
Our observation is a pivotal evidence of the presence of MUTKRAS in cftDNA of tumors with sensitizing MUTEGFR resistant to EGFR-TKIs. A small percentage of our patients received EGFR-TKI as third line therapy and a new biopsy was not repeated at this time. It could be possible that MUTKRAS appeared before the initiation of TKI as a mechanism of resistance to previuos therapy. This hypotesis is weakened by the evidence that patients given second or further lines of therapy showed TTP and OS similar to patients treated in first line, although a mechanism of resistance does not necessarily affect survival. The presence of MUTKRAS has been recently reported using next generation sequencing analysis of tumor re-biopsies after progression under EGFR-TKI treatment , similarly to colo-rectal cancer treated with EGFR antibodies [32, 33]. It remains to be determined if the presence of p.T790MEGFR and MUTKRAS coexist in the same tumor cell or arise in different subclones.
Targeting MUTKRAS proteins is still a challenge [34, 35]. Theoretically, combined treatment with KRAS and EGFR inhibitors can be administered to patients to prevent MUTKRAS-dependent resistance or restore sensitivity to EGFR-TKIs, as recently demonstrated co-targeting EGFR and MEK . To date, p.T790MEGFR remains the most important predictor of efficacy of third generation EGFR-TKIs. Moreover, it was found that the coexistence of both activating MUTEGFR and MUTKRAS was not necessarily a negative predictor for EGFR-TKI therapy . With these evidences in mind, all patients with p.T790MEGFR should receive third generation EGFR-TKI, even in the presence of MUTKRAS. Theoretically, it is possible that MUTKRAS identify a less responsive subgroup of patients but this hypothesis should be validated by monitoring patients prospectically during second-line therapy. Beside p.T790MEGFR and MUTKRAS, other mechanisms of acquired resistance not evaluated in our study have been described in tumor re-biopsies after EGFR-TKI progression, including actionable mutations of MET , HER2 , PIK3CA  or transformation into small cell histology (3%) .
In conclusion, despite the small number of patients involved, the retrospective analysis and the low rate of re-biopsies, our results confirm the importance of cftDNA analysis for the monitoring of secondary mutations associated with EGFR-TKI resistance in NSCLC and underline the role of a highly sensitive approach, i.e., ddPCR, for the detection of low-level mutations. The clinical relevance of these findings, expecially for what concerns MUTKRAS, needs to be evaluated prospectively. These observations open new perspectives in the molecular mechanisms of acquired resistance, indicating a possible role of MUTKRAS in tumor escape from pharmacologic treatment. The effect of MUTKRAS in NSCLC with activating MUTEGFR needs to be further elucidated at the molecular level and encourages the development of inhibitors MUTKRAS for optimal treatment of patients.
A total of 33 NSCLC patients with activating MUTEGFR (exon 19 deletion [ex19delEGFR], exon 21 c.2573T > G [p.L858REGFR] or exon 19 c.2240T > C [p.L747PEGFR]), receiving EGFR-TKI (gefitinib or erlotinib) as per approved indication were included in this study. The analysis of EGFR mutations in primary tumors was performed by standard diagnostic procedures in use in each centre participating to this study (i.e., EGFR TKI response®, Diatech, Jesi, Italy; Therascreen®, Qiagen, Valencia, CA, USA). MUTKRAS were not examined at the time of diagnosis because mutually exclusive with activating MUTEGFR. Plasma and/or re-biopsy samples were taken at the time of disease progression. The analysis of MUTKRAS mutations and EGFR c.2369C > T (p.T790MEGFR) in plasma was not part of standard clinical management and the study was submitted and approved by the Ethics Committee of Pisa University Hospital and conducted in accordance to the principles of the Declaration of Helsinki; all patients gave their signed informed consent before blood collection and cftDNA analysis.
Six ml of blood were collected in EDTA and centrifuged at 4°C for 10 min at 3000 rpm within two hours after blood drawing. Plasma samples were stored at −80°C until analysis. cftDNA was extracted using a QIAmp Circulating nucleic acid Kit (Qiagen®, Valencia, CA, USA) from 1 to 3 ml of plasma following the manufacturer's protocol and the DNA was eluted in 100 μl of buffer.
The investigational part of this study included the assessment of codons 12, 13 (p.G13D) and 61 MUTKRAS and MUTNRAS, p.T790MEGFR, V600EBRAF in cftDNA. Other mutations potentially associated with EGFR-TKI resistance were not examined because of the limited amount of cftDNA available. The analysis of cftDNA was performed by digital droplet PCR (ddPCR, BioRad®, Hercules, CA, USA) and ddPCR Mutation Assay (BioRad®). The analytic procedure was unable to discriminate the nature of the MUTKRAS because the analysis was performed with a ddPCR KRAS Multiplex assay.
PCR reactions were assembled into individual wells of a single-use injection molded cartridge, according to the following protocol: 20 ng of template DNA (4 μl), 1 μl of 20X target primer/probe assay (FAM), 1 μl of 20X wild type primer/probe assay (HEX), 10 μl of 2X ddPCR Super Mix and 4 μl of DNAse/RNAse-free water up to a total volume of 20 μl. Droplet generation oil (70 μl) was then loaded and the cartridge was placed into the droplet generator. Using vacuum, sample and oil were mixed, generating mono-disperse droplets. Thereafter, 40 μl of packed droplets were transferred into a 96-well PCR plate for thermal cycling amplification. The protocol was standardized for all mutations to the following conditions: 95°C × 10 min, 94°C × 30 s and 55°C × 60 s (35 cycles), 98°C × 10 min, and 4°C hold. The droplet reader (BioRad®) was used for fluorescence signal quantification. The concordance between MUTKRAS and p.T790MEGFR was assessed on pairwise cftDNA and tissue DNA of 8 patients who underwent re-biopsy for diagnostic purposes. DNA was extracted from formalin-fixed paraffin-embedded biopsies using the QIAmp DNA Mini Kit (Qiagen®) and analyzed using conventional diagnostics as reported above. As a positive control for MUTKRAS, the cftDNA from 30 patients with known MUTKRAS pancreatic cancer was used, while the DNA extracted from plasma of 43 healthy blood donors was employed as negative control for MUTKRAS and p.T790MEGFR.
TTP and OS were calculated following standard procedures and survival curves were generated by the SPSS statistical software (version 16).
This study was supported in part by grants from the Italian Association for Cancer Research (AIRC, Italy), MIUR and ITT (Italy) and Fondazione Berlucchi (2014) to RD; AIRC (grant #12182), Cam-Pac FP7 (grant #602783) and FIMP-Ministero Salute (CUP_J33G13000210001) to AS.