Background
Personalized medicine becomes increasingly important in cancer treatment. Drugs targeting specific mutated genes or specific activated pathways are clinically available for several indications, e.g. for
EGFR-mutated lung cancer [
1]. Currently, many more targeted therapies are being evaluated in clinical trials and show promising results either alone or in combination with other drugs [
2]. As molecular markers are constantly expanding, predictive analysis should be easily adaptable to future clinical need. In this era of personalized medicine, next generation sequencing (NGS) analysis using gene panels is increasingly becoming a standard diagnostic approach as interrogation of multiple molecular markers is required with only a limited amount of (mostly) formalin-fixed, paraffin-embedded (FFPE) tissue [
3]. These markers include a spectrum of genetic alterations, ranging from small base pair alterations (e.g. point mutations, small deletions, or small insertions) to larger structural variants (e.g. translocations, amplifications, or deletions) affecting genes or large regions of chromosomes. Besides therapy decision, the presence or absence of specific genetic alterations can contribute to the differential diagnosis and provide relevant prognostic and predictive value [
4]. In addition, mismatch repair deficiency, causing instability of repetitive DNA sequences known as microsatellites, was shown to be predictive for response to immune checkpoint blockade for a range of tumor types [
5].
Sequencing is considered a high resolution approach for the detection of small genetic alterations. With the introduction of sensitive NGS techniques, mutations can reliably be detected in a very low number of cells. We have previously implemented and validated the single molecule Molecular Inversion Probes (smMIP) approach that includes molecular tagging to identify PCR duplicates. These duplicate reads are merged into consensus reads, which leads to the elimination of PCR and sequencing artifacts. In combination with the strand-specific analysis to distinguish genuine mutations from deamination artefacts, it is possible to detect point mutations, small deletions, small insertions, and complex mutations (indels) down to at least 1% mutant allele frequency, as well as to reliably exclude sequence variants with a mutant allele frequency > 3% [
6]. In addition, existing gene panels are easily adaptable [
6] and thereby this technology can comply with the growing clinical need. Taken together, the smMIPs technology allows screening of multiple therapeutic targets with high sensitivity using FFPE tissue material in a routine diagnostic setting.
Detection of copy-number variations (CNVs) at specific genomic locations, e.g. amplification of
MET, is frequently performed by fluorescence in situ hybridization (FISH). In addition, several genome-wide approaches are available, including (shallow) whole genome sequencing or (SNP) array. Interestingly, in the last few years it was shown that NGS-based approaches using defined gene panels are able to identify CNVs in parallel to sequence alterations [
7‐
10]. Recommendations were published to guide the detection and reporting of copy number gains using gene-panel NGS data in a routine diagnostic setting [
11].
Recent developments, showing clinical benefit of immune checkpoint blockade in microsatellite instability (MSI) positive tumors, increased the importance of tumor agnostic MSI detection [
5]. MSI is characterized by spontaneous gains or losses of nucleotides in small nucleotide repeat regions (microsatellites) and reflects a state of genomic hypermutability. In diagnostic FFPE tissue, the microsatellite status is routinely investigated by a pentaplex PCR investigating five microsatellites [
12]. This analysis was mainly validated in Lynch syndrome associated tumors, but little data is available regarding the reliability of this analysis in other tumor types [
13,
14]. Studies focusing on the spectrum of unstable microsatellites across multiple cancers show different patterns of MSI among cancer types [
15‐
17]. NGS analyses can be used to study multiple microsatellite loci in one test [
13,
17‐
21].
Taken together, different molecular techniques (i.e. NGS, FISH, and pentaplex PCR) are often used for predictive analyses for cancer therapy. NGS gene panel analysis allows detection of these genomic aberrations in one assay, which will potentially save valuable time, material and costs. Therefore in the current study we aimed to develop a single smMIP-based NGS assay, which can detect sequence alterations, amplifications, and MSI. To comply with the increased number of therapeutic opportunities based on molecular aberrations, both in regular therapeutics and in clinical trial settings, our panel was designed to allow predictive screening for the four most requested molecular-screened indications, i.e. lung cancer, colorectal cancer, melanoma and gastro-intestinal stromal tumors (GIST), but is also useful for evaluation of a broad spectrum of other tumor types as well. Molecular markers for initial therapy decisions as well as markers important for disease progression (i.e. resistance mechanisms) were included in the panel. In addition, we validated and implemented the detection of clinically relevant gene amplifications in compliance with recently published recommendations [
11], and we included the analysis of 55 microsatellites for MSI detection across multiple cancer types.
Discussion
In the current study, a smMIP-based NGS approach was validated, which aimed at concurrent detection of genomic mutations, amplifications, and MSI in small amounts of histological tumor tissue or cytological material representing routinely available tumor samples. We provide detailed information on the validation of the procedure for predictive testing in current clinical practice and show that it is feasible to reliably analyze different types of genomic aberrations using a small smMIP-based NGS panel on diagnostic materials. Based on this sensitive and reliable molecular diagnostic analysis, cancer patients can be stratified for targeted therapies and immunotherapy regarding currently available drugs. The combined analysis of different types of genomic aberrations within one analysis saves valuable time, material, and costs. In addition, a broad predictive analysis allows identification of genomic aberrations that are rarely considered by clinicians, e.g. MSI in a non-MSI-prone tumor or a
KIT mutation in melanoma, which might be beneficial for treatment options [
5,
51,
52].
Previously, simultaneous detection of mutations with either amplifications or MSI using targeted NGS was described [
10,
13,
19,
53‐
55]. To our knowledge this is the first small smMIP-based NGS panel that allows concurrent analysis of mutations, amplifications, and MSI by numerous microsatellite loci. Although whole-genome sequencing offers the most comprehensive analysis, the lack of fresh frozen material, high costs and low amounts of tissues/neoplastic cells hinder its use in routine diagnostics. Targeted NGS approaches on the other hand offer lower costs, can yield higher coverage for regions of interest, offer a fast turnaround time, and allow analysis of FFPE specimens with suboptimal gDNA quality. Our panel uses a PCR-based target enrichment method, which is suitable for small gene panels and can handle low (< 10 ng) gDNA input. Upscaling our routine diagnostic panel from the previously published Cancer Hotspot Panel (247 smMIPs [
6]) to the PATH panel (663 smMIPs) did not affect sensitivity. In addition, the smMIP panel can be easily adapted by adding smMIPs that target new regions to an already optimized smMIP pool [
6], which is an advance in the constantly changing field of personalized medicines and molecular markers, as was demonstrated by the addition of microsatellite markers. Nevertheless, the PATH panel does not prevent additional predictive analyses. Immunohistochemistry of PD-L1, ALK and ROS1, or mRNA based analysis to detect relevant fusion genes involving
ALK,
ROS1,
RET, or
NTRK genes cannot be replaced by the PATH panel [
56]. In addition, tumor mutational burden, associated with a favorable response to immune checkpoint inhibitors [
57,
58] cannot be performed by a small NGS panel. Although large (commercial) NGS panels (> 1 Mb) can be used for this analysis instead of whole-genome sequencing or whole-exome sequencing, these panels are generally more expensive, time consuming (due to the complexity of the analysis and interpretation of results), and not appropriate for all diagnostic requests.
The PATH panel was designed to cover most targetable mutations and amplifications for lung cancer, colorectal cancer, melanoma, and GIST for first line treatment options, as well in the setting of therapy resistance (e.g.
EGFR,
ALK and
KIT gatekeeper mutations). In addition, the panel can be used to evaluate possible treatment options for other cancer types in a named or compassionate use program. The 729 diagnostic tumor samples that were evaluated with the smMIP panel showed 788 (likely) pathogenic mutations, including mutations that give access to targeted treatment options (e.g. mutations in
EGFR,
BRAF,
MET,
ERBB2, KIT, PDGFRA). Several large deletions and insertions in
MET and
KIT were found. Moreover, clinically relevant amplifications were identified, including targetable amplifications (i.e.
MET,
ERBB2,
FGFR1) and amplifications (
MDM2) associated with hyperprogressive disease upon immunotherapy treatment [
33,
44‐
46,
48,
50,
59]. Targetable aberrations were not only detected in lung cancer, colorectal cancer, melanoma and GIST, but also in the other cancer types. Lastly, MSI was observed in both MSI-prone and non-MSI-prone tumor samples, which predicts response to immunotherapy [
5,
51]. These results show that with the PATH panel a broad spectrum of actionable genetic alterations can be evaluated.
During validation, cut-offs for gene amplifications and MSI analysis were established. In addition, we have identified pitfalls of these analyses. Amplification analysis by NGS relies on sequencing coverage possibly combined with VAFs of germline polymorphisms (SNPs) [
11]. We based our analysis on sequencing coverage. Before coverage outliers can be detected, a suitable normalization method has to be applied. The median coverage was shown to be a convenient normalization method, whereas the mean and summed coverage appeared less suitable. To detect coverage outliers, coverage in the sample can be compared to normal samples in the same sequencing run or an external reference pool [
10,
60‐
62]. This latter one saves valuable costs and time (due to lack of availability of a normal sample for every tumor sample) and was shown to generate reliable results. Detection of outliers can be achieved by determining a threshold [
54,
63,
64], calculating a
p-value [
7,
61], or bootstrap based estimation of the confidence intervals [
65]. We chose for the threshold method and validated a relative coverage ≥3.0. This method allows quantification of amplifications by deducing the number of alleles in the tumor cells from the relative coverage and tumor purity of the sample [
11]. Accordingly the clinical relevance of amplifications can be established (i.e. low or high number amplification). In addition, the number of gene copies that needs to be present to be quantifiable by the NGS analysis can be deduced and should be considered to be included in the diagnostic report to specify restrictions of NGS-based detection of copy number gains [
11]. Although these deductions depend on the estimated tumor purity, which is error-prone, the VAF of somatic variants can be used to support this estimation. If a low sensitivity is obtained or low-level amplifications are of interest, one could consider FISH analysis to detect a specific amplification or OncoScan array to comprehensively analyze copy number variations. Despite that these analyses are more sensitive to exclude the presence of amplifications, especially in samples with limited tumor load and to detect low level amplifications or amplifications in a subset of tumor cells with clusters, the smMIP-based NGS based analysis offers a cost-effective multiplex approach.
Like amplification analysis, MSI analysis was established without the need for a paired normal sample. The five markers that are routinely studied by the pentaplex PCR are mainly validated in Lynch syndrome associated tumors. This approach is not necessarily applicable to NGS based analysis on different tumor types. With respect to the tumor type, different microsatellites might be affected. Indeed, studies focusing on the spectrum of unstable microsatellites across multiple cancer types showed different patterns of MSI [
15‐
17]. The NGS based methodology also differs from the fragment length analysis based read out. In the smMIP-based NGS panel 55 microsatellite repeats were included that were reported unstable across multiple cancer types in NGS based analysis [
15,
16]. By increasing the number of microsatellites that are evaluated, the smMIP-based NGS panel may be more sensitive for a broad range of tumor types compared to the traditional pentaplex PCR. If new predictive microsatellite loci are identified in future studies, smMIPs that target these microsatellite regions can be added to the smMIP pool. Tumor purity also contributes to the sensitivity of the assay. We determined a cut-off of at least 30% tumor cells to reliably exclude MSI and a fraction of unstable loci of over 30% to classify a sample as MSI. The relative low number of MSI-high samples in colorectal carcinoma urged additional validation which confirmed the assay sensitivity. The low frequency of MSI in the colorectal carcinoma samples in the diagnostic cohort, is in line with a low frequency of MSI-high tumors among metastasized colorectal cancer cases [
66].
Conclusion
More genomic information can be extracted from tumor samples than ever before, but less material is available for testing. In the current study, we show that reliable, sensitive, and simultaneous detection of mutations, gene amplifications, and MSI can be achieved in routinely available diagnostic tumor samples using an integrated smMIP-based NGS approach, which was designed in close collaboration with clinicians and molecular biologists of several specialized centers. This single smMIP-based NGS test reduces the number of analyses that are typically performed on tumor samples, uses only limited amount of material, and thereby simplifies the workflow for molecular cancer diagnostics. Moreover, the panel allows easy adaptation and can thereby comply with the rapidly expanding molecular markers.
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