Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients


Abstract

Background:

When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA.

Methods:

Our panel (‘SiRe’) covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice.

Results:

SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression.

Conclusions:

SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.

Main

Precision medicine, coupled with the tissue-based assessment of biomarkers predictive of treatment outcome, has transformed pathology practice (Papadopoulos et al, 2006). RAS and BRAF mutation testing in colorectal cancer (CRC; Di Nicolantonio et al, 2008; Lièvre et al, 2008), EGFR in non-small-cell lung cancer (NSCLC; Lynch et al, 2004) BRAF in melanoma (Chapman et al, 2011) and cKIT and PDGFRα in gastrointestinal stromal tumours (GIST; Antonescu, 2008) has added a genotypic element to the phenotypic diagnostics of solid tumours. However, tumour tissue is not always available or may be insufficient for molecular testing, especially when cancer is diagnosed at advanced stages on small biopsy specimens. On other occasions, due to tumour location or small size, tissue sampling can be challenging and risky, particularly in extensively treated patients. As an alternative to cancer tissue, predictive biomarkers can be non-invasively assessed in cell-free DNA (cfDNA; Schwarzenbach et al, 2011; Crowley et al, 2013).

Using a Taqman-derived assay (TDA) we previously identified EGFR mutations in NSCLC (Karachaliou et al, 2015) and BRAF mutations in melanoma patients (Gonzalez-Cao et al, 2015) with a specificity of 100% and with sensitivities of 69% and 78%, respectively. One of the factors contributing to this high sensitivity was the concomitant analysis, in each patient, of serum- and plasma-derived cfDNA (Karachaliou et al, 2015; Gonzalez-Cao et al, 2015). This performance may be further improved by next-generation sequencing (NGS), which can be multiplexed across several genes to cover less common and even novel variants (Malapelle et al, 2016a). Large gene panels or whole-exome approaches to screen for a large number of genomic regions may not be easily implemented in cfDNA analysis (Cancer Genome Atlas Research Network, 2014). Conversely, small NGS panels tailored to target a limited number of actionable genes can be an effective tool in daily clinical practice (Paweletz et al, 2016). This strategy, known as ‘ultra-deep sequencing’, can significantly increase sensitivity, which is essential considering that circulating tumour DNA represents only a small fraction (<0.5%) of the total cfDNA (Mead et al, 2011) in most patients with solid tumours. Since the low threshold levels of mutant alleles required to detect clinically relevant alterations may easily lead to false-positive results (van Dijk et al, 2014), implementation of the ultra-deep sequencing of cfDNA in the clinical setting must be validated in terms of blood collection, cfDNA extraction, automated library preparation, sequencing and variant calling (Gargis et al, 2012; Malapelle et al, 2016c).

In this proof-of-concept study, we report the development, performance evaluation and implementation in a clinical setting of a narrow gene panel that targets 568 clinically relevant mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα) involved in non Small cell lung cancer, gastroIntestinal stromal tumour, metastatic coloRectal carcinoma and mElanoma (whose acronym is SiRe). This panel has a high sensitivity and specificity and enables the detection and quantification of mutations in cfDNA purified from the plasma and serum of patients with different types of solid tumours.

Materials and methods

Design of the SiRe panel

The Ion AmpliSeq Designer suite v5.3.1 with hg19 was used as reference genome to develop a customised panel targeting six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα) that are associated with treatment outcome in NSCLC, GIST, CRC and metastatic melanoma (Lynch et al, 2004; Antonescu, 2008; Di Nicolantonio et al, 2008; Lièvre et al, 2008; Chapman et al, 2011). A single primer pool leading to the selection of 42 amplicons (ranging from 125 to 175 bp) enabled us to cover all COSMIC annotated mutations (n=568) in the selected exons of the target genes. The complete reference range of SiRe is reported in Supplementary Material (Supplementary Table S1). The amplicon design (available on request) covering 5.2 kb of genomic DNA was optimised for the simultaneous analysis of 16 samples with the 316v2 chip (Thermofisher, Foster City, CA, USA) on a Personal Genome Machine Torrent (Thermofisher).

Study design, patients and samples

The panel performance was evaluated in three steps (Figure 1). First, the analytical sensitivity of the assay was assessed on DNA from two cell lines and by using an artificial reference standard with multiple mutations in different genes. Second, clinical sensitivity and specificity was determined using archival cfDNA from 63 cancer patients (Table 1) with paired tumour tissue, previously genotyped with a TDA. As exploratory analysis, to confirm that our NGS approach cover the mutations in cKit and PDGFRα genes, two GIST samples (bloods and tissues) were tested with SiRe and the relative data are reported only in Supplementary Material. Third, the performance of the panel in daily clinical practice was assessed using blood samples prospectively collected from patients with advanced NSCLC. Written informed consent was obtained from all patients and documented in accordance with the general authorisation to process personal data for scientific research purposes from ‘The Italian Data Protection Authority’ (http://www.garanteprivacy.it/web/guest/home/docweb/-/docwebdisplay/export/2485392). All information regarding human material was managed using anonymous numerical codes, and all samples were handled in compliance with the Helsinki Declaration (http://www.wma.net/en/30publications/10policies/b3/).

Figure 1
Figure 1

Study design.cfDNAs (A) extracted with the QIAsymphony virus/pathogen kit (B) from paired (P) plasma and (S) serum (C) samples were analysed by quantitative 5′-nuclease TaqMan PCR (D) and by the NGS SiRe panel (E). Any discordance between the two techniques was evaluated by dPCR (F). After preclinical validation, the SiRe panel was applied in clinical practice in cases in which tissues were not available to select patients for TKI treatment, at baseline (G), and to evaluate the selection of resistant clones after disease progression (H).

Table 1: Characteristics of the patients included in the retrospective (left) and prospective (right) clinical validation of the SiRe panel

DNA purification

DNA from the two cell lines was isolated using the QIAamp Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Circulating-free DNA was purified as follows: 15 ml blood was withdrawn from patients and collected in Vacutainer tubes (BD, Plymouth, UK). Plasma and serum were isolated by centrifugation twice at 2300 r.p.m. for 10 min. The supernatant (serum or plasma) was aliquoted and used immediately for cfDNA isolation or stored at −80 °C. Cell-free DNA was purified from serum and plasma for each patient (1.2 ml). In the rare instances that the volume of the serum and plasma sample obtained from a patient was between 1 and 1.2 ml, PBS up to 1.2 ml was added to the samples, which were then purified using the QIAsymphony robot (Qiagen) and the QIAsymphony DSPVirus/Pathogen Midi Kit, according to the manufacturer’s instructions, and cfDNA was eluted in a final volume of 30 μl. Since correct preanalytical handling of blood specimens is crucial to maintain the sample informative, the process was standardised (in terms of blood collection, sample centrifugation and cfDNA extraction) in the Department of Public Health of the University of Naples Federico II, and all procedures were performed in-house by a nurse belonging to the laboratory staff.

Sample sequencing

We analysed the serum and plasma cfDNAs of each patient enrolled in the study. Libraries were constructed and purified on the Ion Chef (Thermofisher), and eight samples (corresponding to 4 patients) were added per run. Library generation was as follows: 6 μl of cfDNA were dispensed on Ion Code plates and amplified using Ion AmpliSeq DL8 (Thermofisher). We used 22 cycles for cfDNA amplification and 6 cycles for library reamplification after barcoding, under the thermal conditions defined by the manufacturer. Purified libraries derived from eight cfDNA samples were diluted to 60 pM and combined with eight additional cfDNA-derived libraries to obtain a 16 Ion Code pooled library. The two-pooled libraries were re-loaded into the Ion Chef instrument, and templates were prepared using the Ion PGM Hi-Q IC Kit (Thermofisher). Finally, templates were loaded into the 316v2 chip and sequenced on PGM.

Data analysis

Signal processing and base calling were carried out using the default base-caller parameters on Torrent Suite [v.5.0.2] and coverage analysis was performed using SiRe designed bed files with coverage plug-in (v.5.0.2.0). BAM files were visually inspected with the Golden Helix Genome Browser v.2.0.7 (Bozeman,MT, USA). Variants were automatically annotated using variant caller plug-in (v.5.0.2.1) at specific optimised parameters of the SiRe panel (Supplementary Table S2). In particular, only variants with 5X allele coverage and a quality score 20, within an amplicon that covered at least 1000X alleles, were called, and the frequency of each mutant allele was recorded.

Preclinical assessment

Genomic DNA from the HCC827 (EGFR p.E746-A750del; KRAS wt) and A549 (EGFR wt; KRAS p.G12S) cell lines was used to assess analytical performance. Both cell lines were obtained from the National Research Council/Institute of Experimental Endocrinology and Oncology on courtesy of Dr Pierlorenzo Pallante (Naples, Italy). The analytical sensitivity of the assay for point mutation and indel detection was determined by diluting DNA from the appropriate mutated cell line (A549 for point mutations and HCC827 for indels) into increasing concentrations of DNA from the appropriate wt cell line (HCC827 for point mutations and A549 for indels). DNA dilutions ranged between 1 : 10 and 1 : 10 000, which correspond to allelic fractions from 1 : 20 to 1 : 20 000 of the mutated allele (both cell lines are heterozygous). Each dilution was analysed in duplicate to estimate inter-run assay reproducibility, and the library obtained from each dilution was sequenced twice to evaluate intra-run assay reproducibility. In addition, customised Horizon Diagnostics Multiplex gDNA reference standard, with mutation in EGFR (p.E746_A750del and p.G719S), KRAS (p.G12D), NRAS (p.Q61L) and BRAF (p.V600E), each of them at three different dilution points (1, 0.5 and 0.1%), were assessed to provide stronger evidence on SiRe analytical performance.

Clinical validation

We determined the specificity and sensitivity of our assay by analysing archival serum and plasma cfDNA from 40 cancer patients at presentation attending the Quiron Dexeus University Hospital (33 NSCLC, 2 CRC and 5 metastatic melanoma) with paired tumour tissue. In addition, we tested archival serum and plasma cfDNAs from 12 responder patients and 11 patients at the time of tumour progression after treatment (18 NSCLC, 2 CRC and 3 metastatic melanoma; Table 1). All of the 63 cfDNA samples and tumour tissues had previously been genotyped for EGFR, KRAS, NRAS and BRAF mutations using a TDA (Gonzalez-Cao et al, 2015; Karachaliou et al, 2015). In the case of tumour tissues, genotyping had been confirmed by standard PCR followed by Sanger sequencing. Cases showing discordance between the NGS SiRe panel and the TDA were further investigated by digital PCR (dPCR) on a QuantStudio 3D Digital PCR System platform (Thermofisher) as previously described (Malapelle et al, 2016b).

Performance of the SiRe panel in prospective clinical samples

To evaluate the performance of the SiRe panel in the clinical setting, we prospectively genotyped 79 advanced NSCLC patients (37 men and 42 women; mean age: 65 years) using blood samples collected at the Department of Public Health of the University of Naples Federico II. According to the European Medicines Agency guidelines, mutations related to EGFR disease were tested in patients when tissue was not available at presentation (n=46), or at tumour progression (n=33) in patients previously treated with erlotinib (n=14), gefitinib (n=14) or afatinib (n=5) in the attempt to detect the emergence of resistance secondary mutations. In 21 of the 33 cases with tumour progression, first-line TKI administration had been based on the demonstration of an EGFR mutation in tissue, whereas in the remaining 12/33 cases, TKI treatment had been administrated in second line without evidence of EGFR mutations.

Results

Panel design and preclinical performance evaluation

The SiRe panel was designed to cover 568 clinically relevant mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα) involved in NSCLC, GIST, CRC and metastatic melanoma (see Supplementary Table S1). The panel was intended for use in cfDNA purified from patients with advanced cancer. On cell line derived DNA, the SiRe panel detected the EGFR deletion p.E746_A750del and the KRAS point mutation p.G12S at a level as low as one copy of the mutated allele in a background of 20000 copies of wild-type alleles (0.005% mutated allele fraction), with 100% of intra- and inter-run reproducibility. In addition, regarding the results obtained on multiplex gDNA reference standard (Horizon Diagnostics), p.E746_A750del and p.G719S point mutation in EGFR, p.G12D mutation in KRAS exon 2, p.Q61L mutation in NRAS exon 3 and p.V600E mutation in BRAF exon 15 were correctly identified for each different dilution point.

This high analytical performance was achieved thanks to the use of optimised parameters set in variant caller plug-in (v.5.0.2.1) which detected low abundant mutated alleles with a specificity of 100% (see Supplementary Table S2).

Clinical sensitivity and specificity of the SiRe panel in cfDNA samples

The retrospective series of cfDNAs (Supplementary Table S3) was constituted by 126 paired serum and plasma samples from 63 patients. In each run, up to 16 paired serum and plasma samples from eight patients on 316v2 were processed. Run median output was 257Mbases, median read length was 124 bp, mean read depth was 2821 × and coverage uniformity was 97%. Technical performance data relative to each processed sample are reported in Supplementary Material (Supplementary Table S4). When the 63 samples were tested with the SiRe panel, the cfDNA of all eight patients with wild-type tumour tissue was negative (specificity 100%, CI 64.6-100%). In the remaining 55 patients with EGFR, KRAS, NRAS or BRAF mutations in tumour tissue, the SiRe panel detected the same mutation in the serum and/or plasma cfDNA in 46 cases (sensitivity 83.6%, CI 67.3–94.3%; Table 2).

Table 2: Concordance of Taqman-derived assay (TDA) and the SiRe panel NGS in retrospective serum and plasma cfDNA samples

Comparison of the SiRe panel with a TDA in cfDNA samples

We compared the performance of the SiRe panel for mutation analysis in cfDNA with that of a previously reported TDA (Karachaliou et al, 2015; Gonzalez-Cao et al, 2015) in 63 samples: (i) the 40 cfDNA samples obtained at presentation mentioned above; (ii) archival serum and plasma cfDNAs from 12 patients in response to different types of antitumour drugs; and 11 patients mutations in the cfDNA of 46 of 63 patients. The test was positive in both serum and plasma cfDNA in 35 patients (76.1%), positive in plasma but not in serum in 5 patients (10.9%), and positive in serum but not in plasma in 6 patients (13%). An EGFR sensitising mutation and the p.T790M resistance mutation were detected simultaneously in 10 patients at progression to EGFR TKIs.

As reported in Table 2, there was a high concordance (Cohen’s Kappa 0.85) between the results obtained with the NGS SiRe panel and the TDA, although the performance of the SiRe was slightly better. All 42 patients with mutation-positive cfDNA at TDA were also positive with the SiRe panel, and the 17 negative samples with the panel were also negative at TDA. In addition, NGS detected mutations in the cfDNA of four patients, whereas TDA did not. The mutations in these four patients appeared also in paired tumour tissue. One was a p.L597R mutation in BRAF not covered by the TDA, and was confirmed by dPCR (Supplementary Figure S2). The remaining three mutations were a p.L861Q mutation in EGFR and two KRAS mutations, p.G12C and p.G12A. Both TDA and NGS using the SiRe panel enable quantification of the mutated alleles (Figure 2). There was a significant correlation in the levels of serum cfDNA between the two techniques (r=0.64). In contrast, correlation was lower in the case of plasma (r=0.35), but improved significantly when three outlier samples were removed (r=0.61).

Figure 2
Figure 2

Quantification of mutated allele fractions.Comparison of the quantification of mutated allele fractions by Taqman Derived Assay vs SiRe NGS in serum (A) and plasma (B) cfDNA. In the case of plasma, three outliers were removed and results re-plotted (C).

Evaluation of the SiRe panel for prospective analysis of clinical samples

The performance of the SiRe panel in the clinical setting was evaluated by prospectively testing the serum and plasma cfDNA of patients with advanced NSCLC for whom no tissue was available in order to select them for TKI treatment. Seventy-nine patients were tested, 46 at presentation and 33 at the time of tumour progression after first-line TKI treatment (Table 1). The NGS procedure was adequate for variant calling in the 79 cfDNA paired serum and plasma samples. The run metrics parameters were not dissimilar from those of the retrospective samples. In fact, in prospective cfDNA samples, the median output was 210Mbases, the median read length 125.57 bp, the mean read depth 3385.45 and coverage uniformity 97.49%. Among the 46 patients analysed at baseline (Supplementary Table S5), we detected four EGFR mutations (8.7%), one point mutation in exon 18 (p.G719A), two deletions in exon 19 (both p.E746_A750delELREA) and one insertion in exon 20 (p.H773-V774insH). In all four patients, the mutant alleles were detected in both serum and plasma cfDNA and were confirmed by digital PCR (data not shown).

Regarding samples at progression (Supplementary Table S6), the SiRe panel did not detect mutations in 12 patients, whose tissues had been identified as EGFR wild type in biopsies at presentation. In contrast, among the 21 patients EGFR positive in baseline tissue, the SiRe panel confirmed the same mutation in cfDNA in 19 cases (Table 3). Thus, sensitivity and specificity in this cohort of patients at progression were within the range of those observed in the retrospective cohort. Interestingly, in 9 of those 19 cases (47%), we observed the emergence of the EGFR p.T790M mutation in addition to the original EGFR activating mutation. The appearance of EGFR p.T790M mutation in relation to TKIs treatment regimen was reported in Figure 3. Of the 28 mutations (sensitising+p.T790M) detected, 10 (35.70%) were present in both serum and plasma, 7 (25%) in plasma alone and 11 (39.3%) in serum alone. All mutations detected by the SiRe panel at progression were confirmed by dPCR.

Table 3: Comparison of the mutational status in FFPE tumour tissue at presentation with the results of the SiRe panel in archival cfDNA purified from serum and plasma baseline (n=42, left) and at response or after tumour progression (n=23, right)
Figure 3
Figure 3

Frequency of the EGFR p.T790M mutation (green: T790M−; red T790M+) after progression to thyrosine kinase inhibitors (TKIs) in the serum and plasma cfDNA of EGFR-mutated patients evaluated with SiRe panel NGS.A full colour version of this figure is available at the British Journal of Cancer journal online.

Discussion

In this proof-of-concept study, we demonstrate that the performance of ultra-deep sequencing using a narrow NGS panel on Ion Torrent PGM is excellent, and that this procedure can be used for the routine testing of relevant tumour mutations in cfDNA. The high sensitivity (90.5%) and analytical specificity (100%) of this panel equal or even surpass those of such other procedures as real-time PCR-based methods. Unlike earlier NGS applications that cover large genomic regions (Cancer Genome Atlas Research Network, 2014), our small gene panel (5.2 kb) focuses on biomarkers that are currently used in the clinical setting.

The ultra-deep sequencing procedure reported herein has various advantages. In fact, using a single panel, we were able to detect up to 568 relevant mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα). These mutations included less common, but actionable variants such as the BRAF p.L597R mutation in melanoma (case #38 in Supplementary Table S3). Sequencing with the SiRe panel was more efficient than real-time PCR target techniques in detecting deletions (n=2) and point mutations (n=6) on cfDNA samples. In addition, NGS per se is a time-effective procedure for analysing large numbers of samples, thereby optimising the work flow in molecular pathology laboratories (Malapelle et al, 2016a). With our procedure, different types of samples (DNA from tumour tissues and cfDNAs from biological fluids) from patients affected by different types of diseases (e.g., NSCLC, GIST, CRC and melanoma) can be processed simultaneously. Consequently, sample batching is more effective and does not require a minimum number of a given tumour type. As a result, turnaround time (TAT) can be as short as three working days, as recommended by international guidelines (Lindeman et al, 2013). The recently developed Ion Chef automated library preparation station, which has a better procedure reproducibility and standardisation than manual procedures, also contributes to the short TAT (Malapelle et al, 2016a).

The Ion Torrent PGM protocols, panels and variant caller do not detect low abundant mutations diluted in a large amount of WT DNA. Therefore, we used several in-house strategies specifically tailored to cfDNA. Firstly, we reduced the number of genes and exons vs commercially available tests, and we modified the thresholds for variant calling, in particular all the variants with 5X allele coverage and a quality score 20, within an amplicon that covered at least 1000X alleles, were called (Supplementary Table S2).

We also adapted the Ion Chef template preparation protocol by pooling two 16-sample libraries in each run. Thus, using this well standardised procedure, we were able to sequence simultaneously up to 32 paired plasma/serum samples in less than 3 h on the PGM, with a consequent reduction in the total consumable cost. In a previous study (Malapelle et al, 2016a)we showed that by using a commercially available 22 gene panel(AmpliSeq Colon and Lung Cancer Panel)on the Ion Torrent PGM, the consumable cost was €196 per sample. Using the modified protocol that we developed in this current study the cost per sample was lowered to 98 euro for simultaneously analysis of six different genes. This is comparable with the cost of the most commercially available Real Time PCR based kits.

The simultaneous analysis of paired plasma/serum samples is a crucial feature of this new procedure since the sensitivity of somatic mutation analysis in cfDNA increases when serum and plasma are analysed together (Gonzalez-Cao et al, 2015; Karachaliou et al, 2015). Our results are in agreement with this finding. In fact, of the 89 patients found to carry mutations in cfDNA, 58 (65.17%)were positive in both serum and plasma, 15 (16.85%) in plasma alone and 16 (17.98%)in serum alone.

From the technical point of view, even when sequencing 16 samples simultaneously in a run, the SiRe panel had optimal run metrics in our daily clinical practice in terms of both mean depth reads and uniformity of coverage, which resulted in a high assay sensitivity in cfDNA vs tumour tissue (90.5%) and a specificity of 100%. This is a very high degree of concordance, particularly given the 91.7% concordance between paired surgical resection and cytological samples (Sun et al, 2013). Thanks to the high sensitivity of our assay, the EGFR mutational rate of 8.7% that we identified in NSCLC patients prospectively tested on cfDNA at baseline is in keeping with previous data on tissue samples (Malapelle et al, 2013). Similarly, the frequency of the EGFR p.T790M mutation, which was detected in the cfDNA of 9 of 19 (47.4%) patients progressing after TKI treatment (n=5 gefitinib, n=3 afatinib, n=1 erlotinib), is in line with data obtained on tissues samples collected after disease progression (Karachaliou et al, 2015).

The performance of our methodology compares favorably with that of NGS for mutational analysis in the blood of cancer patients. An Ion Torrent-derived sequencing of five genes in cfDNA purified from never smoking lung cancer patients achieved a modest 58% sensitivity and 87% specificity (Couraud et al, 2014). An analysis of 23 amplicons in five genes using cfDNA from breast cancer patients identified 10 mutations but missed 6 identified by droplet digital PCR (Guttery et al, 2015). When restricted to EGFR, deep sequencing achieved 61–80% sensitivity and 94–98% specificity in advanced NSCLC (Uchida et al, 2015). The 90.5% sensitivity of our assay also exceeds the 77% recently reported when NSCLC plasma-derived cfDNA was analysed on an Illumina NGS platform with a panel covering amplicons of 11 clinically relevant genes (Paweletz et al, 2016). Despite the variations inherent to the platforms used, such as the library preparation and the longer TAT (6 days), the Illumina-based NGS approach featured similar run metrics and analytical parameters as our assay, which supports the use of ultra-deep sequencing in the clinical setting (Paweletz et al, 2016). It is conceivable that the higher sensitivity achieved by our panel is due not only to technical differences but also to the simultaneous testing of serum and plasma in each patient.

Besides being an alternative to molecular diagnosis at presentation when tumour tissue is not available, liquid biopsy is also a noninvasive test with which to monitor response to targeted therapy and to detect the emergence of resistance mutations in genes such as EGFR (Sundaresan et al, 2016) and ESR1 (Chu et al, 2016). Monitoring would consist in quantifying the mutant allelic fractions in cfDNA over time, which can be reliably assessed by our NGS assay. The SiRe panel detects the appearance of resistance mutations such as EGFR p.T790M (Figure 3). Finally, the non-synonymous mutation burden correlates with a good response to immunotherapy in NSCLC (Rizvi et al, 2015) and other tumours, and NGS has been proposed as a tool with which to design customised immunotherapies that target common driver mutations (Nielsen et al, 2016). Our panel, which covers several exons in frequently mutated genes, can be useful also in this setting.

In conclusion, we have developed and translate in clinical setting an NGS assay based on a narrow gene panel. The assay detects relevant mutations in cfDNA purified from the serum and plasma of patients with the tumours most commonly tested for molecular alterations (such as NSCLC, CRC and metastatic melanoma). The SiRe panel has excellent sensitivity and specificity, and is hence suitable for testing blood samples in the clinical setting. Finally, it enables the application of NGS on a prospective basis in daily molecular predictive pathology practice, particularly when tumour tissue is not available, and is a tool with which to monitor disease course.

Evaluation of Biomarkers for HER3-targeted Therapies in Cancer


Integration of biomarkers into the majority of drug development programs has led to a need for robust measurements and assay validation techniques for analyses of biological samples. The importance of solid methodologies for biomarker assessment is heightened by the fact that new drugs frequently only offer modest benefit and that many potential biomarkers are continuous variables, the application of which relies on data interpretation, with the risk of subjectivity bias, to establish thresholds. Patritumab is a fully human anti-human epidermal growth factor receptor 3 (HER3) antibody that inhibits HER3 from binding to HRG (Mendell et al., 2015). In the HERALD phase II trial, before data unblinding but after subject enrollment, heregulin (HRG) was prospectively declared to be the predictive biomarker for patritumab efficacy. Advanced non-small cell lung cancer (NSCLC) patients previously treated with at least one chemotherapy regimen were randomized to erlotinib plus patritumab (high- or low-dose) or erlotinib plus placebo (Mendell et al., 2015). Testing a single primary predictive biomarker hypothesis to identify those patients most likely to benefit from patritumab was a secondary objective of the trial and HRG was identified as a continuous biomarker to predict outcome.

Members of the HER family of receptor tyrosine kinases (RTK) and their respective ligands constitute a robust biologic system that plays a key role in the regulation of cell-proliferative growth, survival, and differentiation (Ma et al., 2014). HER3 transactivation via dimerization with other RTKs is frequently observed in various malignancies, including NSCLC. Binding of the alpha and beta forms of neuregulin 1, collectively known as HRG, exposes a dimerization arm in the extracellular domain of HER3 and promotes receptor–receptor interactions (Ma et al., 2014, Carraway et al., 1994). HER3 contains six phosphotyrosine binding sites for the p85 subunit of PI3K, the greatest number of all HER family members, and is a major cause of treatment failure in cancer therapy (Ma et al., 2014, Fedi et al., 1994). Recently, the role of HER3 in primary and acquired resistance to EGFR-targeted or other targeted therapies in NSCLC patients has attracted considerable attention (Ma et al., 2014, Torka et al., 2014). Since HER3 lacks or has weak intrinsic kinase activity, targeting it with blocking antibodies that inhibit HRG binding is one strategy currently being investigated in order to overcome therapeutic resistance (Ma et al., 2014).

In the study by Mendell et al., although no progression-free survival (PFS) benefit was observed overall with the addition of patritumab to erlotinib, when patients were stratified according to HRG mRNA levels HRG-high patients treated with patritumab and erlotinib had significantly improved PFS compared with patients treated with erlotinib alone in both the high- and low-dose arms (Hazard Ratio (HR), 0.37 [95%CI, 0.16–0.85] and 0.29 [95%CI, 0.13–0.66]) (Mendell et al., 2015). No PFS benefit was observed in HRG-low patients. An exploratory analysis suggested that high HRG expression might also be a negative prognostic factor in patients treated with single-agent erlotinib (Mendell et al., 2015).

The role of HRG expression as a marker of HER3 activity has been previously reported. Constitutive activation of HER3 signaling can occur in the absence of direct genetic activation of HER3 or HRG while HER3 activation does not occur as a result of mutation or amplification of the HER3 co-receptors EGFR or HER2. Chronic HER3 signaling is driven by high level and potentially autocrine expression of HRG (Holmes et al., 1992). When HRG and HER3 expressions were profiled in more than 750 patients with head and neck squamous cell carcinoma, high-level expression of HRG was associated with constitutive activation of HER3, defining an actionable biomarker for interventions targeting HER3 (Shames et al., 2013).

Since the arrival of erlotinib and gefitinib, metastatic EGFR positive lung cancer patients can be offered therapeutic alternatives with proven superiority over standard platinum-based chemotherapy (Rosell et al., 2013). Testing for EGFR mutations to guide patient selection for EGFR inhibitors, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, is highly recommended (Lindeman et al., 2013). As commented by Mendell et al., the use of a prospective–retrospective approach applied to a single predictive biomarker hypothesis has the advantage of avoiding a high false-positive rate due to multiple comparisons when multiple biomarker hypotheses are evaluated on an equal footing in an exploratory fashion (Mendell et al., 2015). But are statistical simulations able to dismiss the confounding interactions that EGFR-sensitizing mutations could have on the HRs observed in the study? Some readers may also wonder why, in a study of primarily erlotinib treatment where samples were obtained from most patients, EGFR mutations were not assessed? Clinical trials with EGFR inhibitors designed without using EGFR mutation status, as an enrolment criterion should not be an acceptable practice anymore. Finally, having lost >50% of samples for analyzing HRG mRNA, can we safely conclude that high HRG mRNA and not HER3 expression levels are correlated with patritumab efficacy?

Although technological improvements in terms of specimen acquisition and processing have been made, much work remains to be done to ensure the quality of biospecimens and harmonization of tissue collection, processing and storage procedures, attributable largely to the long-standing success of formalin-fixed paraffin-embedded tissue analysis as the standard in diagnostic pathology. There is an ongoing trend to improve standardization of procedures for biomarker development in oncology that involves academia, professional organizations, and industry. Identification and widespread use of biomarkers will ensure that patients receive the best possible therapeutic strategies, thereby avoiding unnecessary treatments and associated toxicities, and reducing total health costs. Increased awareness of HER3 function in cancer progression and tumor recurrence following drug resistance has several implications for future lines of investigation. High expression of HRG seems to accurately define a population of tumors that may have an oncogenic dependency on ligand-activated signaling via HER3 (Mendell et al., 2015). Based on the results of the Mendell et al. study, a two-part phase III study (NCT02134015) has been initiated to examine patritumab plus erlotinib treatment in EGFR wild-type patients with advanced NSCLC. Part A will enroll subjects with any HRG value to further refine the HRG cutoff level while evaluating the efficacy of patritumab plus erlotinib versus erlotinib in the HRG-high group. Part B will enroll only HRG-high (as per revised criteria) patients to evaluate efficacy and safety of patritumab plus erlotinib versus erlotinib.

Source:http://www.ebiomedicine.com

Genetic Testing in New NSCLC: Worth the Effort?


“If you build it, they will come,” the saying goes, but has that been the case with genetic/genomic testing in patients with newly diagnosed non-small cell lung cancer (NSCLC)? After all, research has shown that about 40% of patients with NSCLC have one of these molecular alterations in their tumor, and using these tests can help sort out those who have KRAS-mutated disease, versus epidermal growth factor receptor (EGFR) mutations, versus ALK, ROS1, or RET translocations, guiding patients to the most effective targeted therapy.

A recent international survey of oncologists revealed that while the use of genetic/genomic is on the rise, many clinicians are either bypassing testing altogether, or if the patient did undergo testing, the results were not used to make treatment decisions.

 Overall, genetic testing can help clinicians get a better handle on the subtype of NSCLC they are contending with, based on specific molecular alterations. This improved disease description will give patients a better chance of receiving the most effective treatment with the least toxicity and, ideally, at the right price point.

For instance, EGFR mutations are currently the most common genomically classified subgroup of NSCLC. These mutations tend to be more prevalent in tumors with adenocarcinoma histology, in patients who have never smoked tobacco, in patients of East Asian race, and women. Results of genetic/genomic testing can then set patients up for treatment with agents such as gefitinib (Iressa) and erlotinib (Tarceva).

Guidelines established by specialty groups, including the College of American Pathologists and the International Association for the Study of Lung Cancer, recommend testing for EGFR mutations (along with testing for ALK mutations) in patients with advanced-stage disease. Whether early-stage patients should undergo the same is uncertain.

“The question of whether or not to test a diagnostic specimen in early-stage disease is a local decision that must be made in conjunction with each institution’s oncology care team, as insufficient published evidence supports a universal recommendation,” according to 2013 guidelines from the groups mentioned above. “The benefits of testing all early-stage disease patients must be balanced against the cost of performing testing that may not be used to select therapy for the patients who never have relapse.”

But genetic screening in early NSCLC isn’t out of the question, wrote Jean-Charles Soria, MD, PhD, of Gustave Roussy Cancer Center in Villejuif, France, in an email to MedPage Today. He pointed out that there is a debate over the risk of relapse in stage I-II cancer, but even if that risk is over a 5-year period, it “reaches 50%, so I also believe it could help.”

 Even in early-stage NSCLC, molecular testing can serve as a decisive factor in the choice of therapeutic strategies for patients, explained Frédérique Nowak, PhD, of the French National Cancer Institute (INCa) in Boulogne-Billancourt, France in an email to MedPage Today.

For example, early testing can rule out if a patient is simply ineligible for treatment with gefitinib, an EGFR tyrosine kinase inhibitor (TKI). Nowak cited a 2012 study that she and Soria co-authored that found that EGFR testing avoided a median of 8 weeks of administration of gefitinib for EGFR-negative patients.

An added benefit to testing? The potential for a significant reduction in treatment costs. “In France in 2010, about 15,000 of the 16,834 patients with lung cancer who benefited from EGFR screening were EGFR-mutation negative and thus were ineligible for TKI-EGFR treatment,” Nowak stated.

“As 8 weeks of gefitinib treatment costs €4,600 per patient in France [about $5,000], this would mean overall savings of €69 million [about $74 million]. According to the numbers of EGFR tests performed in 2011, the spared costs should be even greater,” she added.

Seven years ago, INCa, along with the French Ministry of Health, launched a campaign to implement molecular testing for all cancer patients across the French national healthcare system. The network is made up of 28 regional molecular genetic centers that perform tests for free for all patients in their regions.

 Nowak reported that the network now “is fully deployed in the country. For lung cancer, the tumors of more than 24,000 patients were screened for EGFR mutation and ALK rearrangement in 2014. It roughly corresponds to all non-epidermoid NSCLC patients at a metastatic stage in France.”

Back to the survey results, which revealed that one of the impediments to implementing genetic testing in newly diagnosed patients may be the wait for the results – the turnaround time for test findings can range from 1 to 2 weeks. About a quarter of U.S.-based survey respondents stating that was too long.

In general, lung cancer takes some time to develop before a diagnosis is made; a few extra days of waiting for test results that can have a major impact on treatment course and patient outcomes shouldn’t be that onerous, noted survey leader James Spicer, PhD, MBBS, of King’s College London.

“Personally — and I think this is a fairly common viewpoint — turnaround within 1 week — 5 working days — would definitely be acceptable; 2 weeks or more becomes a problem,” he said.

While Spicer told MedPage Today that he wasn’t surprised by the survey results, his group also did not find any evidence that clinicians were skeptical about EGFR testing overall. Instead, it may be a matter of continual education to emphasize the benefits of genetic testing in NSCLC patients across the board.

“We should be aiming for every suitable NSCLC patient to be tested, and every patient receiving an appropriate treatment for their type of lung cancer,” Spicer said in a written statement about the survey results. He added that “there is still work to be done in emphasizing the importance of obtaining EGFR test results prior to the initiation of treatment, and using this vital information to select optimum therapy.”

How to manage AEs associated with EGFR TKIs, ALK inhibitors among NSCLC patients


Compared with standard chemotherapy, EGFR tyrosine kinase inhibitors (TKIs) and anaplastic lymphoma kinase (ALK) inhibitors are associated with improved efficacy in patients with EGFR-mutant and ALK-rearranged non-small cell lung cancer (NSCLC). However, these agents are not without adverse events (AEs), says a leading oncologist at the IASLC Asia Pacific Lung Cancer Conference (APLCC) 2016 held recently in Chiang Mai, Thailand.

“Both EGFR TKIs and ALK inhibitors are generally less toxic than chemotherapy,” said Professor Caicun Zhou, director of the Department of Oncology, Shanghai Pulmonary Hospital and chairman of the Oncology Department of Tongji University in Shanghai, China., “However, these TKIs are associated with a number of bothersome AEs that need to be managed carefully.”

Improper management of AEs could affect tolerability and efficacy of therapies as duration of targeted therapies with progression-free survival of about 1 year is longer than chemotherapy, he added.

Diarrhoea, for example, induced by EGFR TKIs, requires good patient evaluation, diet modification and standard dose loperamide, said Zhou. If diarrhoea is resolved within 12 hours, loperamide should be discontinued and the diet adjusted. For persistent diarrhoea (grade 1/2), loperamide should be increased to 4 mg and 2 mg every 2 hours thereafter. Severe diarrhoea (grade 3/4) that comes with fever, dehydration and blood in stool however requires hospital admission, octreotide administration, intravenous fluids, and antibiotics as needed. “Laboratory studies are also required as well as titration of octreotide dose upward as necessary,” added Zhou. [Curr Oncol 2011;18:126-138]

The risk factors for diarrhoea include female sex, low body surface area, comorbidities, renal failure, and elderly age. “If patient is to be started on afatinib, prophylaxis with antidiarrhoeal diet – no spicy and greasy foods – is recommended,” said Zhou. Milk is also avoided, so are cabbage, Brussel sprouts and broccoli as these are difficult to digest. [Future Oncol 2015;11:267-277]

The presence of paronychia in some patients requires temporary discontinuation of EGFR-TKI for 2-4 weeks. Upon improvement to grade 1, EGFR TKI may be reintroduced, said Zhou. Dose is dependent on clinician discretion. “If toxicities do not worsen, we usually escalate the dose.”

If paronychia does not improve, EGFR-TKI should be discontinued and clobetasol cream 2-3 times daily should be started as needed, he added.

Professor Caicun Zhou

Professor Caicun Zhou

Interstitial lung disease (ILD) or interstitial pneumonitis associated with EGFR TKIs may also present in NSCLC patients, requiring physicians’ careful review of patient history, risk factors, respiratory signs and symptoms, and chemotherapy or radiation. “If your patient develops ILD, it is crucial to discontinue the drug and initiate high-dose steroids, coupled with oxygen therapy or mechanical ventilation as supportive treatment,” said Zhou.

Third generation EGFR-TKIs meanwhile showed striking efficacy against T790M mediated acquired resistance, with minimal efficacy against WT EGFR and low rate of skin rashes, he added.

“We have also good data to show that second generation ALK inhibitors can successfully overcome acquired resistance to crizotinib and are well-tolerated,” Zhou said.

“Overall, alectinib and ceritinib were both well-tolerated, with low proportions of patients needing dose reduction, interruption and withdrawal. The most common AEs reported with alectinib were constipation, fatigue and peripheral oedema and most were grade 1 to 2.”

Circulating tumour DNA helps detect EGFR mutations


While biopsy remains the gold standard for EGFR mutation testing in patients with advanced non-small-cell lung cancer (NSCLC), circulating tumour-derived DNA (ctDNA) may provide a more feasible methodology, according to studies presented at the European Lung Cancer Conference (ELCC) 2015 held in Geneva, Switzerland.

“We were looking for a valid test that can identify an EGFR mutation when the tumour is not accessible for bronchoscopy or CT-guided biopsy, and that’s in agreement with the gold standard tissue test,” said Dr. Martin Reck from the Lung Clinic Grosshansdorf, Germany.

Reck reported data from the real-world ASSESS study, which compared tumour biopsy with plasma ctDNA in 1,162 matching samples from European and Japanese patients. “Mutation status showed a high 89 percent concordance between the two methods,” he said. “The sensitivity of the plasma test was 46 percent, specificity was 97 percent, and positive predictive value [PPV] 78 percent.” [ELCC 2015, abstract 35O_PR]

He noted that use of a highly sensitive DNA sequencing methodology and identical methods for tissue and plasma testing in a subset of patients further increased sensitivity to 72 percent, specificity to 99 percent and PPV to 94 percent.

“While improvements are still required in mutation analysis practices of both tissue/cytology and plasma samples, our data show that plasma ctDNA may be a feasible, suitable sample for EGFR mutation analysis,” he suggested. “It is important to use robust and sensitive methodologies to ensure patients receive appropriate treatment to address the molecular features of their disease.”

Another study reported the extraction of urine ctDNA to test for EGFR T790M mutation — a hallmark of disease progression in advanced NSCLC that is useful for patient monitoring. [ELCC 2015, abstract 36O]

The investigators obtained urine samples from patients who progressed on erlotinib and were confirmed to have EGFR T790M mutation by a tumour biopsy test. “EGFR T790M status was analyzed by a sensitive assay that had a lower limit of detection of 2 copies in a background of 20,000 copies of wild-type DNA,” explained Dr. Hatim Husain from the University of California, San Diego, CA, US.

Using this assay, they detected T790M mutation in 10 out of 10 confirmed EGFR T790M-positive patients (sensitivity, 100 percent). In addition, three patients with negative tissue testing results tested positive by urine analysis. EGFR T790M mutation was detected as early as 3.5 months prior to radiographic progression on first-line EGFR tyrosine kinase inhibitor (TKI) therapy, identifying five patients who may be eligible for second-line EGFR TKI treatment due to emergence of T790M mutation.

“This method, combining the extraction of urine ctDNA with an ultra-sensitive next-generation sequencing and mutation enrichment technology, has the advantage of urine as ctDNA source, potentially enabling dynamic monitoring of EGFR TKI therapy response from a completely noninvasive sample,” concluded Husain.

EGFR Testing Not Done in 25% of Lung Cancer Patients


Many patients with advanced non-small cell lung cancer (NSCLC) are missing out on a crucial genetic test to determine whether they would benefit from more targeted therapy, new research suggests.

A survey of more than 550 oncologists around the world revealed that EGFR genetic testing is not being conducted in about 25% of patients with NSCLC. And even when the test is requested, treatment is started before the results are back in one-quarter of cases.

The research was presented at the 2015 European Lung Cancer Conference in Geneva.

Guidelines from the International Association for the Study of Lung Cancer (IASLC) recommend that all patients diagnosed with advanced NSCLC, except those with squamous cell carcinoma, undergo EGFR testing, and that the results be used to guide treatment decisions.

Recent research has indicated that matching EGFR mutation status to specific tyrosine kinase treatments can improve overall survival.

However, the IASLC guidelines are not being followed in a number of cases, said lead researcher James Spicer, MD, PhD, reader in experimental oncology at King’s College London at Guy’s Hospital in the United Kingdom.
“Not only were some suitable patients not tested at all for tumor EGFR mutations, some patients did undergo testing but the treatment decision about whether to give an EGFR inhibitor or chemotherapy as first-line treatment was taken without reference to the result,” he said in a statement.

A representative sample of 562 oncologists from Canada, France, Germany, Italy, Japan, South Korea, Spain, Taiwan, the United Kingdom, and the United States completed an online survey from December 2014 to January 2015.

The survey assessed the prevalence of mutation testing, attitudes and barriers to testing, and the way the results affect the choice of therapy for patients with advanced NSCLC.

Dr Spicer and colleagues found that EGFR testing was requested before the administration of first-line therapy for 81% of patients with stage IIIb/IV NSCLC. The most common reason for not ordering a test was that the patient had the wrong type of histology.

“Most centers, especially the big academic centers, know enough about those patients not to waste resources on testing for a mutation that they are very unlikely to find,” Dr Spicer explained.

“Sure enough, the most likely reason that our respondents cited for not testing was that patients have the wrong type of tumor to have a mutation…and that’s fine,” he told Medscape Medical News.

The next most common reason was a lack of diagnostic material. The tumor is difficult to access, so “by the time we’ve decided that it’s a lung cancer in the pathology lab, there isn’t enough cellular material left to extract the DNA and do this sort of genetic testing,” he pointed out.

“But that should be less and less of a problem in the modern era, because we getting better at obtaining biopsies with newer techniques, and we’re getting better at doing the test with more sensitive genetic tests,” he added.

The survey revealed a number of other reasons for not ordering EGFR testing, which Dr Spicer described as “a bit more worrisome.” These included cases in which a patient was deemed unfit to undergo testing.

Not testing someone who is wheelchair-bound or breathless, and thereby not offering the most appropriate treatment, which could quickly improve a patient’s health status, “is really not good modern oncology,” he noted.

“Maybe there’s an area of education around prescribers that still needs to be done,” he added.

Two other reasons cited for not offering EGFR testing — patients were smokers or were too old — go against the guidelines. Many smokers do undergo EGFR mutation, and elderly patients can tolerate, and might benefit from, tyrosine kinase therapy, Dr Spicer noted.

Finally, a major reason cited for not ordering EGFR testing was the long turnaround time. Dr Spicer said he can “sympathize”; the turnaround time is weeks at some centers.

For patients who underwent testing, the oncologist received the results before first-line therapy was initiated in 77% of cases (ranging from 51% in France to 89% Japan). In 23% of cases, first-line therapy was initiated before the test results were available.

“It’s a bit paradoxical,” Dr Spicer said, “to go to the trouble of doing the test and starting the patient on treatment before the mutation test is back.”

“Because these targeted drugs are only licensed by the regulators in the presence of a mutation, if you’re starting before the mutation result is back, then it’s presumably not the targeted drug that you’re starting them on,” he said.

Respondents reported initiating therapy before EGFR results were available because the test took too long or because the patient wanted to start therapy immediately.

According to the survey, 23% of oncologists do not consider EGFR mutation subtypes when making their treatment decisions, but 49% do.

When choosing a first-line therapy for NSCLC patients, a clinically relevant increase in overall survival was cited by 75% of oncologists.

“Overall, it looks like people are getting it right,” Dr Spicer said. However, “there are some significant areas where we could, perhaps, improve things by offering more patients the test and then doing more with that result.”

Dacomitinib as First-Line Treatment in Selected Advanced NSCLC


TAKE-HOME MESSAGE

ABSTRACT

Epirubicin, oxaliplatin, and capecitabine with or without panitumumab for patients with previously untreated advanced oesophagogastric cancer (REAL3): a randomised, open-label phase 3 trial.


Background

EGFR overexpression occurs in 27—55% of oesophagogastric adenocarcinomas, and correlates with poor prognosis. We aimed to assess addition of the anti-EGFR antibody panitumumab to epirubicin, oxaliplatin, and capecitabine (EOC) in patients with advanced oesophagogastric adenocarcinoma.

Methods

In this randomised, open-label phase 3 trial (REAL3), we enrolled patients with untreated, metastatic, or locally advanced oesophagogastric adenocarcinoma at 63 centres (tertiary referral centres, teaching hospitals, and district general hospitals) in the UK. Eligible patients were randomly allocated (1:1) to receive up to eight 21-day cycles of open-label EOC (epirubicin 50 mg/m2 and oxaliplatin 130 mg/m2 on day 1 and capecitabine 1250 mg/m2 per day on days 1—21) or modified-dose EOC plus panitumumab (mEOC+P; epirubicin 50 mg/m2 and oxaliplatin 100 mg/m2 on day 1, capecitabine 1000 mg/m2 per day on days 1—21, and panitumumab 9 mg/kg on day 1). Randomisation was blocked and stratified for centre region, extent of disease, and performance status. The primary endpoint was overall survival in the intention-to-treat population. We assessed safety in all patients who received at least one dose of study drug. After a preplanned independent data monitoring committee review in October, 2011, trial recruitment was halted and panitumumab withdrawn. Data for patients on treatment were censored at this timepoint. This study is registered with ClinicalTrials.gov, number NCT00824785.

Findings

Between June 2, 2008, and Oct 17, 2011, we enrolled 553 eligible patients. Median overall survival in 275 patients allocated EOC was 11·3 months (95% CI 9·6—13·0) compared with 8·8 months (7·7—9·8) in 278 patients allocated mEOC+P (hazard ratio [HR] 1·37, 95% CI 1·07—1·76; p=0·013). mEOC+P was associated with increased incidence of grade 3—4 diarrhoea (48 [17%] of 276 patients allocated mEOC+P vs 29 [11%] of 266 patients allocated EOC), rash (29 [11%] vs two [1%]), mucositis (14 [5%] vs none), and hypomagnesaemia (13 [5%] vs none) but reduced incidence of haematological toxicity (grade ≥3 neutropenia 35 [13%] vs 74 [28%]).

Interpretation

Addition of panitumumab to EOC chemotherapy does not increase overall survival and cannot be recommended for use in an unselected population with advanced oesophagogastric adenocarcinoma.

Discussion

The REAL3 trial is one of two concurrent randomised phase 3 trials (the other being the EXPAND trial15) assessing the addition of anti-EGFR monoclonal antibodies to chemotherapy in first-line oesophagogastric cancer. Based on the findings of REAL3, use of panitumumab in combination with EOC cannot be recommended in an unselected population with advanced oesophagogastric adenocarcinoma, and was associated with inferior overall survival and PFS. Notably, this detrimental outcome in the experimental group was not predicted by the phase 2 endpoint of response rate (overall response rate 52% with mEOC+P). This trial does, however, confirm the efficacy of the EOC control group in this setting, with median overall survival and PFS results that are consistent with those previously reported in REAL2 (11·2 months for overall survival and 7·0 months for PFS).3

The poor outcome associated with mEOC+P in this trial did not seem to be attributable to increased treatment-related deaths, and therefore other potential explanations for our findings need to be considered. First, as reported previously,12combination of panitumumab with full-dose EOC in the initial stages of the trial was associated with unacceptably high rates of grade 3 diarrhoea (four of the first five patients by cycle four). Therefore, we had to reduce the starting doses of oxaliplatin (by 23%) and capecitabine (by 20%) in the experimental group. Although these changes undoubtedly reduced the frequency of grade 3—4 diarrhoea with mEOC+P (17% in phase 3 population), they also served to reduce the dose intensity of chemotherapy, which is reflected in the reduced incidence of grade 3—4 neutropenia and peripheral neuropathy noted in the mEOC+P group. Additionally, the dose intensity data show a reduced proportion of patients achieving at least 80% of the planned capecitabine dose in the experimental group, suggesting that mEOC+P was still slightly more difficult to deliver than standard EOC.

Second, a negative interaction might have occurred between panitumumab and one or more of the EOC components. Evidence in the setting of colorectal cancer suggests that the chemotherapy partner for anti-EGFR therapy might be an important determinant of treatment efficacy, with oxaliplatin-containing regimens yielding inconsistent results. The OPUS16and PRIME11 studies provide evidence of improved outcomes with the addition of cetuximab and panitumumab respectively, whereas no benefit was associated with the addition of cetuximab in the COIN17 and NORDIC VII18 studies in the same setting. Recent cell-line data also suggest that greater synergy might exist between anti-EGFR therapy and irinotecan than with oxaliplatin.19 Additionally, the COIN trial17 results suggest that there might be a differential benefit from cetuximab dependent on the fluoropyrimidine partner, with patients receiving oxaliplatin plus fluorouracil seemingly deriving increased benefit compared with those treated with oxaliplatin plus capecitabine. At present, the significance of these potential interactions is unknown, and has not been assessed in the setting of oesophagogastric cancer.

Third, our findings might have been affected because we assessed panitumumab therapy in a molecularly unselected population. During the years since the inception of the REAL3 trial, several studies have advanced our understanding of the EGFR signalling pathway and its role in oesophagogastric adenocarcinoma. Hot-spot mutations in key oncogenic drivers such as KRAS (common in colorectal cancer) and BRAF (common in malignant melanoma) are now known to be infrequent molecular events in oesophagogastric adenocarcinoma. Indeed, the 5·7% frequency of KRAS mutation in our population is in keeping with the 3—10% reported in other studies,20—22 and we did not note any BRAF mutations in 167 tumour samples tested. By contrast, gene copy number alterations (amplifications and deletions) seem to be a relatively frequent finding in oesophagogastric adenocarcinoma and are more likely to represent the key molecular alterations driving carcinogenesis. Two recent series2324 of detailed genomic analyses in oesophagogastric adenocarcinoma reported that about 37% of tumours harbour copy number aberrations in genes that are deemed to be targetable, including KRASEGFRHER2, and MET. Randomised clinical trials are therefore needed to establish whether targeting of these oncogenic signal transduction pathways can meaningfully improve outcomes for patients.

In preclinical studies, cetuximab can decrease EGFR pathway signalling via reduced phosphorylation of EGFR and AKT in oesophagogastric cancer cell lines.25 In combination with chemotherapy, cetuximab results in synergistic inhibition of cell proliferation and enhanced apoptosis.25—27 In hypoxic gastric cancer cell lines the addition of anti-EGFR therapy reversed oxaliplatin resistance.26 Additionally, a synergistic antitumour effect of combined cetuximab and S-1 was apparent in gastric cancer cell lines overexpressing EGFR.2527 In colorectal cancer, somatic mutations in codon 12, 13, or 61 of the KRASoncogene confer resistance to panitumumab therapy.1128 MET amplification with or without KRAS mutations might be associated with resistance to cetuximab therapy in gastric cancer cell lines;29 however, no validated predictive biomarkers for this setting exist.

Unfortunately, despite preclinical data suggesting a role for anti-EGFR therapy in the treatment of oesophagogastric adenocarcinoma, the REAL3 trial findings are supported by two other phase 3 trials assessing anti-EGFR therapy in this disease setting. The EXPAND trial15 assessed the addition of cetuximab to a cisplatin-capecitabine doublet in 904 patients with previously untreated adenocarcinoma of the stomach and gastro-oesophageal junction, and did not meet its primary endpoint of improved PFS (HR 1·09, 95% CI 0·92—1·29, p=0·32).15 EXPAND also noted no improvement with the addition of cetuximab in either overall survival (HR 1·00, 95% CI 0·87—1·17, p=0·95) or overall response rate (30% in the experimental group vs 29% for controls). The COG trial30 assessed the anti-EGFR tyrosine-kinase inhibitor gefitinib compared with placebo in the second-line treatment of 450 patients with oesophageal and type I—II gastro-oesophageal junction cancers. This trial also did not meet its primary endpoint, with no improvement in overall survival (HR 0·90, p=0·285). However, improvements in PFS (HR 0·795, p=0·017) and disease control at 8 weeks (25·5% in the experimental group vs 16·0% in controls, p=0·014) were noted, suggesting some activity of gefitinib in a small undefined subset of patients.

Taken together, these relatively consistent findings suggest that the EGFR pathway is unlikely to represent an important therapeutic target in most patients with oesophagogastric cancer (panel). The presented biomarker analyses accompanying the REAL3 trial are restricted by small patient numbers and low rates of tested mutations. However, this work is ongoing in the full trial dataset and these translational analyses represent a unique opportunity to further assess the molecular biology of advanced oesophagogastric adenocarcinoma within a randomised trial setting. Techniques such as gene-expression profiling and next-generation sequencing might help to provide further information regarding the driver genetic events in this complex disease. Furthermore, the evaluation of genetic aberrations in pathways linked to EGFR signalling could still offer the prospect of identification of a low-frequency biomarker that identifies a subpopulation of patients benefiting from anti-EGFR targeted therapy in this setting.

Source: lancet