BRAF

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

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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).

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

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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)
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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.

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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.

The immune-related role of BRAF in melanoma

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Abstract

Background
The existence of a dichotomy between immunologically active and quiescent tumor phenotypes has been recently recognized in several types of cancer. The activation of a Th1 type of immune signature has been shown to confer better prognosis and likelihood to respond to immunotherapy. However, whether such dichotomy depends on the genetic make-up of individual cancers is not known yet. BRAF and NRAS mutations are commonly acquired during melanoma progression. Here we explored the role of BRAF and NRAS mutations in influencing the immune phenotype based on a classification previously identified by our group.

Methods
One-hundred-thirteen melanoma metastases underwent microarray analysis and BRAF and NRAS genotyping. Allele-specific PCR was also performed in order to exclude low-frequency mutations.

Results
Comparison between BRAF and NRAS mutant versus wild type samples identified mostly constituents or regulators of MAPK and related pathways. When testing gene lists discriminative of BRAF, NRAS and MAPK alterations, we found that 112 BRAF-specific transcripts were able to distinguish the two immune-related phenotypes already described in melanoma, with the poor phenotype associated mostly with BRAF mutation. Noteworthy, such association was stronger in samples displaying low BRAF mRNA expression. However, when testing NRAS mutations, we were not able to find the same association.

Conclusion
This study suggests that BRAF mutation-related specific transcripts associate with a poor phenotype in melanoma and provide a nest for further investigation.

Has targeted therapy for melanoma made chemotherapy obsolete?

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Targeted therapy for BRAF-mutant melanoma has revolutionised the management of patients with metastatic melanoma1, 2and ignited a quest for even more effective strategies. Situated just downstream of BRAF is MEK, which has become a second molecular target of great interest. The MEK inhibitor trametinib was recently approved by the US Food and Drug Administration (FDA), and other MEK inhibitors are in clinical development.3, 4 Targeted drugs have pushed cytotoxic chemotherapy research into the background, yet chemotherapy regimens are still offered as first-line therapy in countries where access to targeted drugs is limited. Chemotherapy has a deservedly poor reputation in the treatment of advanced melanoma, but it is now valid to ask whether targeted therapy has made chemotherapy obsolete, or alternatively whether combination with a molecularly targeted agent could breathe new life into old drugs.

Caroline Robert and colleagues5 evaluated the combination of dacarbazine with selumetinib, another MEK inhibitor, in a randomised, placebo-controlled phase 2 trial in 91 patients with BRAF-mutant melanoma. The trial’s primary endpoint of improved overall survival was not met. There were, however, signs of clinical benefit for the combination, with a higher confirmed response rate (29% vs 13%) and longer median progression-free survival (5·6 months, 80% CI 4·9—5·9, vs 3·0 months, 2·8—4·6; hazard ratio 0·63, 80% CI 0·47—0·84, one-sided p=0·021) in the selumetinib plus dacarbazine group compared with the placebo plus dacarbazine group. This benefit came at the cost of increased toxicity in the combination group, including rash, oedema, visual disturbances, bleeding, and infection.

The clinical efficacy of the selumetinib-dacarbazine combination was not impressive by the standards now set by BRAF inhibitors. Furthermore, the dacarbazine response rate in the current study (13%)5 was higher than in other recent phase 3 studies,1, 2, 4 which might have inflated results in the combination group as well. Perhaps selumetinib is not the right partner? In other phase 2 trials of selumetinib, the confirmed response rate was 7—11% in patients with BRAF-mutant melanoma.6 Although the combination of selumetinib plus dacarbazine appears to be somewhat better than selumetinib alone, the activity of selumetinib seems less than that reported for trametinib alone (confirmed response rate 22% in the METRIC study).4 Hence, the combination of chemotherapy with a more potent MEK inhibitor could provide superior results.

At the present time selective BRAF inhibitors remain the targeted treatment of choice for metastatic melanoma harbouringBRAF Val600Glu and Lys mutations. The combination of BRAF and MEK inhibitors in patients with BRAF Val600 mutant melanoma is an emerging strategy with early signs of improved clinical benefit.7 Upfront use of concurrent BRAF and MEK inhibition, rather than the addition of a MEK inhibitor after progression on single-agent BRAF inhibitor, seems important.8For patients who do progress after single-agent BRAF inhibitor therapy, the possibility that combination of chemotherapy with a MEK inhibitor might be better than the to-date disappointing results of MEK inhibitors alone deserves further investigation.

Where else might we find a role for MEK inhibitors alone or in combination with chemotherapy as treatment for metastatic melanoma? In patients with this disease whose tumours do not harbour a BRAF Val600 mutation, standard treatment options remain immunotherapy and chemotherapy. There could be a niche for MEK inhibitors with or without chemotherapy in patients not eligible for or refractory to immunotherapy, particularly where their melanomas manifest an upregulated MAP kinase pathway. Indeed, clinical activity of MEK inhibitors has been reported in patients with BRAF Leu597Ser and NRASmutant melanoma, neither of which would be expected to respond to BRAF inhibitor therapy.3, 9 Furthermore, the recently presented results of the phase 2 trial of selumetinib versus temozolomide in uveal melanoma, in which GNAQ or GNA11mutations were identified in 84% of patients, showed superior benefit for patients receiving selumetinib.10 These findings included an objective response rate of 15% in the selumetinib group compared with no responses in the temozolomide group, and more than double the median progression-free survival with selumetinib.

Undoubtedly, MEK inhibitors have a role in the treatment of metastatic melanoma. Although single-agent trametinib is approved by the FDA for front-line use in BRAF Val600 mutant melanoma, MEK inhibitors are likely to become a key element in treatment of melanoma with MAP kinase pathway activation due to oncogenes other than the BRAF Val600 mutation. The combination of a MEK inhibitor with chemotherapy might yet prove to be an effective approach in selected patient populations, such as uveal melanoma, based on the additive benefit of selumetinib and dacarbazine seen in the current study by Robert and colleagues.5 Although treatment paradigms are strongly shifting away from chemotherapy, there might still be an important part for cytotoxic drugs to play in melanoma.

Source: Lancet Oncology

Vemurafenib and Radiosensitization.

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Importance  The BRAF inhibitor, vemurafenib, was recently approved for the treatment of patients with BRAFV600 metastatic melanoma. Wider use of this drug and longer follow-up periods of treatment are resulting in the emergence of a growing number of reports detailing new adverse effects. Cutaneous adverse effects are preeminent with UV-A–dependent phototoxicity, hyperkeratotic folliculitis, hand-foot skin reaction, hair changes, verrucous papillomas, keratoacanthomas, and squamous cell carcinomas.

Observations  We report 2 cases of dermatitis occurring on a previously irradiated skin area in patients treated with vemurafenib for a BRAFV600-mutated metastatic melanoma. The first case occurred 10 days after a low dose of radiation was delivered that usually does not induce any radiodermatitis, suggesting radiosensitization by vemurafenib. The second case occurred 30 days after radiotherapy and was diagnosed as radiation recall dermatitis.

Conclusions and Relevance  Vemurafenib should be considered a potential cutaneous radiosensitizer and an inducer of radiation recall dermatitis. However, these adverse effects are easily managed with topical corticosteroids. Dose reduction or interruption of vemurafenib is not required. Further studies and reports will enlighten us as to whether this pharmacodynamic interaction between x-rays and vemurafenib is also seen with other BRAF or MEK inhibitors on the same mitogen-activated protein kinase pathway currently under development.

Source: JAMA

 

MAP kinase signaling and inhibition in melanoma..

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The mitogen-activated protein kinase (MAPK) pathway is critical to oncogenic signaling in the majority of patients with malignant melanoma. Driver mutations in both NRAS and BRAF have important implications for prognosis and treatment. The development of inhibitors to mediators of the MAPK pathway, including those to CRAF, BRAF, and MEK, has led to major advances in the treatment of patients with melanoma. In particular, the selective BRAF inhibitor vemurafenib has been shown to improve overall survival in patients with tumors harboring BRAF mutations. However, the duration of benefit is limited in many patients and highlights the need for understanding the limitations of therapy in order to devise more effective strategies. MEK inhibitors have proven to particularly active in BRAF mutant melanomas also. Emerging knowledge about mechanisms of resistance as well as a more complete understanding of the biology of MAPK pathway signaling provides insight into rational combination regimens and sequences of molecularly targeted therapies.

Source: Oncozene

 

When to biopsy: A taller-than-wide nodule lacking worrisome sonographic features .

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Two patients were sent to the endocrine clinic for the evaluation of a thyroid nodule. A 67-year-old female was sent for an asymptomatic 1-cm thyroid nodule found during an MRI for neck pain. A 28-year-old female was referred because of a 2-cm thyroid nodule felt during an initial evaluation for infertility.

They had no history of head and neck radiation and no family history of thyroid disease, including thyroid cancer.

The patients were unaware of the thyroid nodule and had no symptoms of dysphagia, change in voice or anterior neck pressure. The 1-cm nodule could not be felt, whereas the larger 2-cm nodule was easily palpable in the left lobe. The nodule was mobile and nontender. Neither woman had palpable adenopathy in the neck. Laboratory testing showed both women had normal thyroid-stimulating hormone values <1.9 mU/L.

IMGANLY_1

Stephanie L. Lee

Both women had a thyroid ultrasound, including a nodal survey of the neck. There were no abnormal or enlarged nodes seen in the bilateral neck in levels 2, 3, 4, 5 or 6.

Nodule features

The older woman had a heterogeneous, minimally hypoechoic/isoechoic 0.9 cm x 0.6 cm x 0.3 cm (sagittal x anteroposterior x transverse) nodule in the left lobe of an otherwise normal thyroid (Figures 1A and 1B). This nodule had a shape that is taller than wide, defined as an anteroposterior/transverse diameter (A/T) ratio >1 in the transverse view. This nodule had an A/T ratio of 2 with a border that was blurred, suggesting varying thickness. There were a few nonshadowing hyperechoic foci that were not punctate and appeared to be posterior to microcytic areas of the nodule. The nodule did not have any vascular flow by Doppler (grade 1) and no calcification.

The younger woman had a mostly isoechoic/hyperechoic 2.9 cm x 2.4 cm x 2 cm (sagittal x anteroposterior x transverse) nodule in the left lobe of an otherwise normal-appearing thyroid (Figures 1C and 1D). The margins of this nodule were well defined. The nodule had an A/T ratio of 1.2 with peripheral and low-volume intranodular vascular flow (grade 3) by Doppler and no calcification. This nodule also contained nonshadowing hyperechoic foci that were not punctate and were posterior to microcytic areas of the nodule.

Characteristics of concern

The generally accepted sonographic characteristics of thyroid nodules that are concerning for malignancy include solid composition, hypoechogenicity, intranodular vascular flow, micro- and macro-calcification, blurred margins, capsular invasion and hypermetabolic rate on F-18 fluorodeoxyglucose (FDG) PET scan.

The shape of a nodule is also independently associated with the risk for thyroid cancer. Cappelli and colleagues found that an A/T ratio >1 was associated with thyroid cancer with a sensitivity of 84% and a specificity of 82%. The taller-than-wide (A/T >1) shape plus two additional worrisome sonographic features (hypoechoic, micro-calcifications, blurred margins and increased intranodular vascular flow) will detect thyroid cancer with a sensitivity of 99% but a specificity of 57%. This group has suggested that biopsy should be performed when a nodule is found with this characteristic on ultrasound. For our patients, the older woman had a <1 cm nodule with a blurred margin and A/T >1 but no other risk factors such as radiation, adenopathy or family history of thyroid cancer. It was elected to watch.

Patient follow-up

After 18 months, the nodule grew in largest dimension to 1.4 cm and developed micro-calcifications. The nodule was biopsied with ultrasound guidance and found to be a papillary thyroid carcinoma. After thyroidectomy, she was found with a unifocal 1.2-cm papillary thyroid carcinoma with classical papillary histology, no invasion, no nodes and BRAF mutation negative. She was not treated with radioactive iodine. She is disease-free after 3 years.

The younger patient had an ultrasound-guided biopsy at the time of the initial visit and was found to have a papillary thyroid carcinoma. After thyroidectomy, she was found to have a unifocal 2.5-cm papillary thyroid carcinoma with classic histology, minimal capsular invasion, two metastatic perithyroidal nodes and BRAF mutation positive. She received 50 mCi radioactive iodine remnant ablation with a post-therapy whole-body scan showing only a small thyroid remnant. She is disease-free after 2 years.

Ultrasound characteristics of these two cases emphasize that thyroid malignancy occurs in nodules that do not have the usual worrisome characteristics of hypoechogenicity, vigorous intranodular vascularity or calcification. Both of these nodules were isoechoic to hyperechoic but had the characteristic of being taller than wide. It is important that clinicians include this characteristic of A/T ratio >1 in the transverse view when describing the ultrasound appearance of a nodule and use it in the decision to biopsy that nodule, regardless of the lack of other sonographic features of malignancy.

References:
  • Cappelli C. Clin Endocrinol. 2005;63:689-693.
  • Cappelli C. Eur J Endocrinol. 2006;155:27-31.
  • Kim EK. AJR Am J Roentgenol. 2002;178:687-691.

·         Source: Endocrine Today.

Drug Combination More Effective than Single Drug for Advanced Melanoma.

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The combination of two targeted drugs—dabrafenib and trametinib—may delay the progression of advanced melanoma longer than dabrafenib alone, a new study suggests. These results, presented September 29 at the 2012 European Society for Medical Oncology Congress (ESMO) and published concurrently in the New England Journal of Medicine, add to a growing body of evidence that combinations of targeted drugs for melanoma are more effective and less toxic than a single targeted drug.

Dabrafenib and trametinib target different parts of a cell signaling pathway altered in melanoma by a mutation called BRAF V600. Single drugs that block BRAF activity shrink melanoma, but the tumors inevitably develop resistance. Researchers hoped that adding a second drug with a different target would slow the development of resistance and disease progression.

In the first part of the randomized phase II trial, which enrolled 85 patients, the researchers determined the combination’s safety and the doses to be used in the trial. The researchers then randomly assigned 162 patients to one of three treatment groups: dabrafenib alone, dabrafenib plus a low dose of trametinib, or dabrafenib plus a higher dose of trametinib. Patients whose disease progressed with dabrafenib alone were allowed to add the higher dose of trametinib to their treatment.

Patients receiving the higher dose of trametinib plus dabrafenib had a median progression-free survival of 9.4 months compared with 5.8 months for patients receiving dabrafenib alone. After 1 year of follow-up, 41 percent of patients receiving the higher-dose of trametinib plus dabrafenib had no disease progression, compared with 9 percent of those who received dabrafenib alone.

Overall, 79 percent of patients in the higher-dose combination group were alive after 1 year, explained Dr. Georgina Long of the Melanoma Institute Australia, one of the study’s authors, at an ESMO press conference. “We have never, ever seen a 12-month survival of that level in metastatic melanoma to date,” she said.

Side effects differed among the treatment groups, and patients in all arms of the trial frequently required temporary or permanent dose reductions. More patients receiving dabrafenib alone (19 percent) developed a secondary squamous-cell skin cancer than patients receiving the higher-dose combination (7 percent), though this difference was not statistically significant. Most patients in the higher-dose combination group (71 percent) experienced a fever compared with a minority of those receiving dabrafenib alone (26 percent).

The trial was funded by GlaxoSmithKline, the drugs’ manufacturer. Two company-sponsored phase III trials of the drug combination are currently under way (here and here).

  • Source: NCI.
  • ”

Health Care Costs a Major Barrier for Young Adult Cancer Survivors

Many young adult cancer survivors do not seek routine medical care because of cost concerns, according to a new study. Even after accounting for health insurance status, survivors of adolescent and young adult (AYA) cancers were much more likely to forgo care in the prior year because of cost concerns, researchers reported September 24 in Cancer.

The results point to potentially serious consequences for AYA cancer survivors, the authors explained. “Medical care in the years following cancer therapy is particularly important to screen survivors for late effects, such as secondary cancers, infertility, and cardiac conditions,” they wrote.

Although the greater availability of health insurance for young adults as a result of the Affordable Care Act will help, they continued, the study indicates “that improvements in post-treatment health care access must be prioritized for this population.”

The study focused on long-term survivors diagnosed and treated between the ages of 15 and 34.

To conduct the study, the researchers used 2009 data from the Centers for Disease Control and Prevention’s Behavioral Risk Factor Surveillance System, a nationwide, state-based system of health surveys conducted each month by telephone. The study included responses from 979 AYA cancer survivors between the ages of 20 and 39 who were at least 5 years past their cancer diagnosis (case subjects) and approximately 67,000 people in the same age range who did not have a history of cancer (control subjects).

Overall, 34 percent of survivors reported forgoing routine care because of cost, compared with 20 percent of control subjects. The groups most affected by cost concerns were survivors between the ages of 20 and 29 and female survivors, Dr. Anne Kirchhoff of the Huntsman Cancer Institute in Utah and her colleagues reported.

AYA survivors also reported being in poor or fair health more often (27 percent versus 9 percent for control subjects) and experiencing frequent mental or physical distress. Forty percent of survivors had not had a routine medical visit in the past year, and 22 percent did not have a personal medical care provider.

Source: NCI.

 

Tumor microenvironment helps skin cancer cells resist drug treatment .

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Neighboring non-cancer cells may contribute to drug resistance

One of cancer’s most frightening characteristics is its ability to return after treatment. In the case of many forms of cancer, including the skin cancer known as melanoma, tailored drugs can eradicate cancer cells in the lab, but often produce only partial, temporary responses in patients. One of the burning questions in the field of cancer research has been and remains: how does cancer evade drug treatment?

New research by a team from Dana-Farber Cancer Institute, the Broad Institute and Massachusetts General Hospital suggests that some of the answers to this question do not lie in cancer cells themselves. To find the answers, scientists are looking beyond tumor cells, studying the interplay between cancer cells and their healthy counterparts. The research team has found that normal cells that reside within the tumor, part of the tumor microenvironment, may supply factors that help cancer cells grow and survive despite the presence of anti-cancer drugs. These findings appear online this week in a paper published in Nature.

“Historically, researchers would go to great lengths to pluck out tumor cells from a sample and discard the rest of the tissue,” said senior author Todd Golub, MD, the Charles A. Dana Investigator in Human Cancer Genetics at Dana-Farber Cancer Institute, pediatric oncologist at Dana-Farber/Children’s Hospital Cancer Center, and director of the Broad’s Cancer Program. Golub is also a professor at Harvard Medical School and an investigator at Howard Hughes Medical Institute. “But what we’re finding now is that those non-tumor cells that make up the microenvironment may be an important source of drug resistance.”

To investigate how the tumor microenvironment may contribute to drug resistance, the researchers designed experiments in which cancer cells were grown in the same wells (miniscule test tubes no larger than a pencil eraser) along with normal cells. These co-cultured cells were then treated with anti-cancer drugs. When grown alone, such cancer cells died in the presence of many of these targeted agents, but when grown together with normal cells, cancer cells developed resistance to more than half of the 23 agents tested.

These observations reflect what clinicians often see in patients with cancers such as melanoma. In the case of melanoma, targeted therapies have been developed against a specific, common mutation in a gene known as BRAF. While some patients’ tumors show an overwhelming response to BRAF inhibitors and seem to disappear, other patients’ tumors only respond by slightly decreasing in size. The failure to shrink tumors at the outset suggests that those tumors possess some level of innate resistance — the ability to evade drugs from the beginning of treatment.

“Even though recent advanced in targeted therapy have caused tremendous excitement in melanoma, the fact remains that drug resistance eventually develops in nearly all metastatic melanomas treated with RAF inhibitors, and in some cases is present at the outset of treatment,” said Levi A. Garraway, MD, PhD, an associate professor at Dana-Farber and Harvard Medical School and a senior associate member of the Broad Institute. “There are many different types of mechanisms that tumors may hijack to circumvent the effects of therapy…no single experimental approach can capture all of these potential mechanisms. Thus, the application of complementary approaches can offer considerable synergy in terms of discovering the full spectrum of clinically relevant resistance mechanisms.”

Scientists have uncovered resistance mechanisms that cancer cells develop over time – genetic changes in specific genes that may give cancer the ability to overcome the effects of a drug with time – but these acquired resistance mechanisms do not explain the innate resistance seen in many tumors.

“We can take cancer cells out of a melanoma patient, put them on a dish, and most times they will turn out to be extremely sensitive to the targeted agents, but that’s not what we see in patients,” said Ravid Straussman, MD, PhD, a postdoctoral fellow at the Broad Institute and first author of the Nature paper. “Why do we get just a partial response in most patients? We set out to dissect this question, and the next logical step was to think beyond cancer cells.”

After completing systematic, high-throughput screens of more than 40 cancer cell lines, the researchers chose to focus on melanoma, looking at whether factors normal cells secrete help cancer cells resist treatment. They measured more than 500 secreted factors and found that the factor most closely linked to BRAF inhibitor drug resistance was hepatocyte growth factor (HGF). HGF interacts with the MET receptor, abnormal activation of which has been tied to tumor growth in previous studies but never to drug resistance in melanoma.

In addition to studying cells in the lab, the research team sought to replicate their findings in samples from cancer patients. Keith Flaherty, MD, director of developmental therapeutics at Massachusetts General Hospital Cancer Center and an associate professor at Harvard Medical School, and his lab provided 34 patient samples for study. The team measured levels of HGF in these samples and saw a relationship between how much HGF was present and the amount of tumor shrinkage patients experienced. For example, tumors in patients with high levels of HGF shrank less than those in patients with low HGF levels.

“To try to explore in patient samples what factors in the microenvironment are not only present but functionally important in drug resistance would have been largely impossible. Coming up with candidates in the lab and then exploring relevance in humans in a targeted way is the only tractable approach,” said Flaherty. “By taking this high-throughput screening, hypothesis-generating approach, we could then follow up by looking at patient samples. In a case like melanoma, where you already have a targeted therapy available, it puts you on good footing to narrow in on specific factors that may be at play in drug resistance.”

Several HGF/MET inhibitors are in clinical development or are FDA-approved for other indications, making clinical trials combining these inhibitors with BRAF inhibitors feasible in the future. In addition, researchers could follow the same approach taken by the team to screen other drugs currently in development, identifying mechanisms of resistance and ways to counter them even before treatment begins.

“Drug resistance should no longer surprise us,” said Golub. “We’re thinking about how to do this – how to systematically dissect resistance – much earlier in the drug development process so that by the time a new drug enters the clinic, we have a good sense of what the likely mechanisms of resistance will be and have a strategy to combat them.”

Source: Dana Faber cancer Institute.