EGFR mutations in lung cancer: from tissue testing to liquid biopsy.

ABSTRACT The presence of EGFR mutations predicts the sensitivity to EGF receptor (EGFR)-tyrosine kinase inhibitors in a molecularly defined subset of non-small-cell lung carcinoma (NSCLC) patients. For this reason, EGFR testing of NSCLC is required to provide personalized treatment options and better outcomes for NSCLC patients. As surgery specimens are not available in the majority of NSCLC, other currently available DNA sources are small biopsies and cytological samples, providing however limited and low-quality material. In order to address this issue, the use of surrogate sources of DNA, such as blood, serum and plasma samples, which often contains circulating free tumor DNA or circulating tumor cells, is emerging as a new strategy for tumor genotyping.

Molecular diagnostics and personalized medicine in oncology: challenges and opportunities.

Increasing evidence demonstrates that target-based agents are active only in molecularly selected populations of patients. Therefore, the identification of predictive biomarkers has become mandatory to improve the clinical development of these novel drugs. Mutations of the epidermal growth factor receptor (EGFR) or rearrangements of the ALK gene in non-small-cell lung cancer, and BRAF mutations in melanoma are clear examples of driver mutations and predictive biomarkers of response to treatment with specific inhibitors. Predictive biomarkers might also identify subgroups of patients that are not likely to respond to specific drugs, as shown for KRAS mutations and anti-EGFR monoclonal antibodies in colorectal carcinoma. The discovery of novel driver molecular alterations and the availability of drugs capable to selectively block such oncogenic mechanisms are leading to a rapid increase in the number of putative biomarkers that need to be assessed in each single patient. In this respect, two different approaches are being developed to introduce a comprehensive molecular characterization in clinical practice: high throughput genotyping platforms, which allow the detection of recognized genetic aberrations in clinical samples, and next generation sequencing that can provide information on all the different types of cancer-causing alterations. The introduction of these techniques in clinical practice will increase the possibility to identify molecular targets in each individual patient, and will also allow to follow the molecular evolution of the disease during the treatment. By using these approaches, the development of personalized medicine for patients with cancer will finally become possible.

Genomic Profiling of KRAS/NRAS/BRAF/PIK3CA Wild-Type Metastatic Colorectal Cancer Patients Reveals Novel Mutations in Genes Potentially Associated with Resistance to Anti-EGFR Agents.

Previous findings suggest that metastatic colorectal carcinoma (mCRC) patients with KRAS/NRAS/BRAF/PIK3CA wild-type (quadruple-wt) tumors are highly sensitive to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs). However, additional molecular alterations might be involved in the de novo resistance to these drugs. We performed a comprehensive molecular profiling of 21 quadruple-wt tumors from mCRC patients enrolled in the "Cetuximab After Progression in KRAS wild-type colorectal cancer patients" (CAPRI-GOIM) trial of first line FOLFIRI plus cetuximab. Tumor samples were analyzed with a targeted sequencing panel covering single nucleotide variants (SNVs), insertions/deletions (Indels), copy number variations (CNVs), and gene fusions in 143 cancer-related genes. The analysis revealed in all 21 patients the presence of at least one SNV/Indel and in 10/21 cases (48%) the presence of at least one CNV. Furthermore, 17/21 (81%) patients had co-existing SNVs/Indels in different genes. Quadruple-wt mCRC from patients with the shorter progression free survival (PFS) were enriched with peculiar genetic alterations in KRAS, FBXW7, MAP2K1, and NF1 genes as compared with patients with longer PFS. These data suggest that a wide genetic profiling of quadruple-wt mCRC patients might help to identify novel markers of de novo resistance to anti-EGFR MoAbs.

Measuring tumor mutation burden in non-small cell lung cancer: tissue versus liquid biopsy.

The introduction in the clinic of immune checkpoint inhibitors (IOs) has represented an important improvement for the treatment of patients with advanced non-small cell lung cancer (NSCLC). These drugs have shown a higher activity as compared with chemotherapy in both first- and second-line of treatment, with some patients experiencing a long-lasting response. More recently, combinations of IOs have entered clinical trials in different tumor types including NSCLC. Nevertheless, IOs are active only in a subgroup of patients and biomarkers for appropriate patients' selection are urgently needed to offer the patients an effective therapy, and also to manage the costs. Tumor mutation burden (TMB) has powerfully emerged as a potential biomarker for immunotherapy and might enter the clinic in the next months, although different challenges are still unsolved. Different methods exist to evaluate TMB in tissue, ranging from whole exome sequencing (WES) to targeted sequencing of smaller sets of genes, which need to be fully standardized to ensure that patients receive an appropriate TMB test with clear clinical interpretation. In addition, as already happened for the implementation of liquid biopsy testing from NSCLC patients to identify targetable alterations, researchers are also evaluating the possibility to calculate TMB in blood, to further enlarge the number of NSCLC patients who may benefit from immunotherapy. Preliminary data highlight the difficulty to develop targeted sequencing panels for the assessment of TMB starting from the circulating cell free DNA (cfDNA). The applicability of TMB testing on liquid biopsy needs further investigation and may be clarified within the ongoing clinical trials.

Targeting the EGFR T790M mutation in non-small-cell lung cancer.

INTRODUCTION: The presence of activating mutations of the epidermal growth factor receptor (EGFR) is predictive of response to first- and second-generation tyrosine kinase inhibitors (TKIs) in patients with advanced non-small-cell lung cancer (NSCLC). However, patients that initially respond to these drugs inexorably become resistant. The T790M mutation in the exon 20 of the EGFR is the main mechanism of resistance to EGFR TKIs occurring in over 50% of the cases. Third generation EGFR TKIs have been shown to be active in patients who progressed after TKI treatment and carry the T790M mutation. Areas covered: This review is focused on the implications of tumor heterogeneity for targeting the T790M in patients with NSCLC. Expert opinion: Pre-clinical and clinical data suggest that the T790M is heterogeneously expressed in tumors that become resistant to first- and second-generation EGFR TKIs. These findings have important implications for the molecular diagnostic of the T790M mutation. Indeed, the analysis of both the circulating free tumor DNA (ctDNA) isolated from plasma and the tumor tissue might provide complimentary information to identify patients carrying the T790M mutation. However, further studies are needed to better understand the influence of tumor heterogeneity on the activity of drugs targeting the T790M.

BRAF V600E mutation in metastatic colorectal cancer: Methods of detection and correlation with clinical and pathologic features.

The screening for BRAF V600E mutation is employed in clinical practice for its prognostic and potentially predictive role in patients with metastatic colorectal carcinoma (mCRC). Little information is available on the sensitivity and specificity of the testing methods to detect this mutation in CRC. By using serial dilution of BRAF mutant DNA with wild type DNA, we found that the sensitivity of allelic discrimination-Real Time PCR was higher than PCR-Sequencing (10% vs 20%). In agreement, the Real Time PCR assay displayed increased analytical sensitivity in detecting the BRAF V600E mutation as compared with PCR-Sequencing in a cohort of 510 consecutive CRCs (21 vs 16 cases). Targeted resequencing demonstrated that all cases negative by PCR-Sequencing had an allelic frequency of the BRAF mutation <20%, thus suggesting tumor heterogeneity. The association of BRAF mutations with clinical and pathological features was assessed next in a cohort of 840 KRAS exon 2 wild type CRC patients screened with the Real Time PCR assay. The BRAF V600E mutation frequency in this cohort was 7.8% that increased to 33.4% in females over 70 y of age with right-sided tumor location. BRAF mutations were also detected in 4.4% of male patients with left-sided tumors and aged <70 y. Fourteen of 61 (22.9%) BRAF V600E mutation bearing patients exhibited microsatellite instability (MSI) as assessed by T17 mononucleotide sequence within intron 8 of HSP110. Our study indicates that Real Time PCR-based assays are more sensitive than PCR-Sequencing to detect the BRAF V600E mutation in CRC and that BRAF mutations screening should not be restricted to selected patients on the basis of the clinical-pathological characteristics.

The S492R EGFR ectodomain mutation is never detected in KRAS wild-type colorectal carcinoma before exposure to EGFR monoclonal antibodies.

The activity of the epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab in metastatic colorectal carcinoma (mCRC) is significantly limited by molecular mechanisms leading to intrinsic or acquired resistance. The S492R mutation of the EGFR, which is caused by either the 1476C>A or the 1474A>C substitution, interferes with binding to cetuximab but not to panitumumab, and has been detected in mCRC with acquired resistance to cetuximab. Since mechanisms of acquired and intrinsic resistance to EGFR monoclonal antibodies in CRC significantly overlap, we evaluated the frequency of the S492R mutation in a series of KRAS-exon 2 wild-type CRC patients. Genomic DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues that were obtained from 505 systemic therapy-naïve CRC patients. A PCR/sequencing method for the detection of the S492R mutation was developed, by using as positive control a plasmid in which the 1474A>C mutation was generated by site directed mutagenesis. The lowest level of detection of this assay was approximately 10% mutant DNA in a background of wild-type DNA. PCR sequencing analysis revealed no S492R mutations in any of the analyzed 505 CRC specimens. Our findings suggest that the S492R mutation is not involved in primary resistance to cetuximab in CRC. Therefore, patients with mCRC should not be routinely screened for this mutation prior therapy with cetuximab.

Detection of EGFR mutations by TaqMan mutation detection assays powered by competitive allele-specific TaqMan PCR technology.

Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct = 37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR.

Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting for the choice of the most appropriate therapy. Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) is a novel modification of the conventional PCR method that selectively amplifies minority alleles from a mixture of wild-type and mutant sequences irrespective of the mutation type or position within the sequence. In this study, we compared the sensitivity of a COLD-PCR method with conventional PCR/sequencing and the real-time PCR-based Therascreen kit to detect KRAS mutations. By using dilutions of KRAS mutant DNA in wild-type DNA from colon cancer cell lines with known KRAS status, we found that Fast COLD-PCR is more sensitive than the conventional PCR method, showing a sensitivity of 2.5% in detecting G>A and G>T mutations. The detection of G>C transversions was not improved by either Fast COLD-PCR or Full COLD-PCR. We next analyzed by COLD-PCR, conventional PCR and Therascreen 52 formalin-fixed paraffin-embedded samples from mCRC patients. Among 36 samples with >30% tumor cells, 8 samples were negative by conventional PCR, Therascreen and Fast COLD-PCR; 20 mutations identified by conventional PCR were confirmed by Therascreen and Fast COLD-PCR; 8 cases undetermined by conventional PCR were all confirmed to carry KRAS G>A or G>T mutations by using either Therascreen or Fast COLD-PCR. Conventional PCR was able to detect only 2 KRAS mutations among 16 samples with <30% tumor cells (12.5%), whereas Therascreen and Fast COLD-PCR identified 6 mutants (37.5%). These data suggest that Fast COLD-PCR has a higher clinical sensitivity as compared with conventional PCR in detecting G>C to A>T changes in the KRAS gene, which represent >90% of the mutations of this oncogene in CRC.