Keywords

DNA library :

Collection of DNA fragments that are captured, barcoded, and clonally amplified, prior to sequencing on NGS platforms.

Gene panel:

Representative gene regions covered by a sequencing assay.

Laboratory developed tests:

Test designed, developed, and adapted in-house after validation.

Limit of detection:

Corresponds to the analytical sensitivity of a given NGS assay, reflecting the lowest amount of analyte which can be reliably detected.

Molecular cytopathology:

Discipline of cytopathology based on the integration of morphologic changes with the genomic alterations/molecular features underlying the development, progression, and prognosis of neoplastic diseases.

Next-generation sequencing:

High-throughput molecular platform that allows sequencing multiple gene sequences in parallel and interrogating various genetic alterations for multiple patients in a single run.

Personalized medicine:

Cancer therapy based on the specific molecular alterations of a patient’s tumor.

Pyrosequencing:

“sequencing by synthesis”-based technology, in which the sequential incorporation of nucleotides is identified by the detection of a released pyrophosphate.

Reads:

DNA fragments that are sequenced by a NGS platform during a run.

Real-Time PCR:

PCR-based assay that detects and quantifies in “real time” the amplification of a given DNA target using specific fluorescent probes.

Reference range:

The interval between the upper and lower concentrations of analyte in the sample for which a suitable level of precision, accuracy, and linearity has been demonstrated

Sanger sequencing:

Standard sequencing technology based on the incorporation of chain-terminating dideoxynucleotides (usually fluorochrome labeled) during the process of sequencing by DNA polymerase.

Turnaround time:

Time required to analyze a sample and deliver a test result from when the sample is accessioned in the laboratory.

Validation:

Procedure that defines the performance parameters of an assay, such as sensitivity, specificity, accuracy, precision, detection limit, range, and limits of quantitation of a novel methodology prior to clinical implementation.

FormalPara Key Points

  • Molecular cytopathology plays a key role in clinical diagnostics, prognostication, and the selection of patients for targeted treatment

  • Modern cytopathologists need to be familiar with molecular techniques to appropriately triage specimens for molecular testing

  • In comparison to histologic material, cytology specimens often provide better-quality DNA

  • Most DNA-based assays including NGS can be successfully applied to cytology specimens

  • In order to improve NGS laboratory workflow, it is important to create a gene panel to cover relevant hotspot targets with a defined cost

  • In-house validation of each new diagnostic methodology implemented in routine practice is required, even when commercially available and validated for in vitro diagnostic use

In the last 10 years, the landscape of Personalized Medicine has included the contribution of Molecular Cytopathology, in particular for advanced stage patients with solid tumors. Since these patients are not candidates for surgical resection, a concurrent histology specimen is not always available [1, 2]. Therefore, in order to be a knowledgeable partner in diagnostic and predictive approaches to cancer therapy, the modern cytopathologist needs to be familiar with the basic principles and some of the more advanced molecular techniques used in clinical practice [3, 4].

Cytology samples (Fig. 5.1) provide high-quality DNA, sufficient for a wide array of DNA-based sequencing assays, including next-generation sequencing (NGS) [5]. This novel high-throughput technology represents an evolution of conventional DNA sequencing methodologies, such as Sanger sequencing and pyrosequencing.

Figure 5.1
figure 1

Examples of different cytologic preparations commonly used for molecular assays: (A) cell block; (B) direct smear; (C) liquid-based cytology; (D) cytospin

Full size image

Sanger sequencing has long been the gold standard for the identification of point mutations, deletions, and small insertions [6, 7]. In this method, a chemically modified nucleotide (dideoxynucleotide) terminates the extension of the DNA strand at the point of incorporation. This results in a mixture of DNA fragments of varying lengths. Each dideoxynucleotide, (A, T, C, or G) is labeled with a different fluorescent dye (dye terminator). The newly synthesized and labeled DNA fragments are sequentially separated by size through capillary gel electrophoresis. The fluorescence is detected by an automated sequence analyzer, and the order of nucleotides (base calling) in the target DNA is visualized as a sequence electropherogram [7, 8]. Although Sanger sequencing was the method first employed in most clinical pathology laboratories, its low sensitivity (around 20% of mutant alleles) limits its application in low tumor content samples, in which the tumor often constitutes a minority of the mixed cell population present. Thus, Sanger sequencing frequently requires tumor enrichment by microdissection prior to analysis to avoid false-negative results [9]. Although low throughput, Sanger sequencing is a robust technology, suitable for analyzing complex genomic regions featuring combined deletion and insertions (Table 5.1).

Table 5.1 Sanger sequencing: principal advantages and disadvantages
Full size table

Pyrosequencing is another method of DNA sequencing by synthesis and is a valid alternative to Sanger sequencing. It relies on the detection of a pyrophosphate released during the DNA polymerase reaction with an enzymatic cascade resulting in the production of visible light [10]. This is converted in analog signal as a peak in a pyrogram. Pyrosequencing provides higher sensitivity (around 5% of mutated allele) than Sanger sequencing, but its error rate (1.07%) is not negligible [11]. When a heterozygous mutation is identified by direct sequencing or by pyrosequencing, both mutant and wild-type alleles are seen on the sequencing electropherograms and on the pyrograms, respectively [10, 11].

With the increase in the number of predictive and prognostic biomarker testing needed for patient management, there is a growing need for high-throughput sequencing technology with the capability of evaluating multiple genes simultaneously. A suitable and flexible multigene testing approach to evaluating known somatic point mutations is by the Sequenom MassARRAY®. This genotyping platform is based on the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and can provide customized genotyping assays to analyze allele-specific primer extension products [12]. The basic principle underlying this assay is that mutant and wild-type alleles for a given point mutation produce single-allele base extension reaction products of a mass that is specific to the sequence of the product. Mutation calls are based on the mass differences between the wild-type product and the mutant products as resolved by MALDI-TOF mass spectrometry [12].

Compared to conventional sequencing technologies, next-generation sequencing (NGS) offers high analytic sensitivity together with a high clinical sensitivity. Analytic sensitivity (also known as allelic fraction) is defined as the ability of a mutational assay to identify an alteration in a background of wild-type alleles. Clinical sensitivity covers the spectrum of possible alterations that can be identified by any given assay [13]. NGS exploits a massively parallel sequencing technology, which increases sequencing throughput from hundreds of thousands to millions of sequences (reads) and enables simultaneous analyses of different gene targets for multiple patients in each run [14, 15]. The balance between analytic and clinical sensitivity seen in NGS, together with the minimal amounts of input DNA required, makes this technology ideal for application in cytology samples. The increasing use of NGS in combination with advanced tumor sampling techniques using novel bronchoscopic/endoscopic approaches makes the practice of cytopathology an attractive field in the realm of molecular medicine [16, 17]. A key advantage of NGS over more targeted sequencing technologies is the opportunity to evaluate biomarkers in novel genes of potential clinical interest, in addition to standard of care testing, and thereby facilitate enrollment of patients in clinical trials [18,19,20].

The principal advantages and limitations of NGS are listed in Table 5.2 [5, 15, 18,19,20,21,22,23,24].

Table 5.2 Next-generation sequencing: principal advantages and disadvantages
Full size table

A variety of NGS platforms are available for clinical use. Despite the availability of different platforms, the NGS workflow is characterized by four principal steps: (1) DNA library generation, (2) single fragment clonal amplification, (3) massive parallel sequencing, and (4) data analysis [16, 21, 25] (Fig. 5.2).

Figure 5.2
figure 2

Schematic representation of the four steps of the NGS workflow, including DNA library preparation, single fragment clonal amplification, massive parallel sequencing, and data analysis

Full size image

The DNA input required to generate the library is dependent on the target gene selection. The Illumina(™) platforms (San Diego, CA, USA) utilize a hybridization-based capture system and require a DNA input ranging from 50 to 250 ng and 24–72 h for processing the sequencing data [22]. Recent advances in library preparation have enabled a reduction in the required input DNA, and Illumina validated protocols can be optimized to analyze 10–100 ng of DNA [26]. Alternatively, the IonTorrent platforms(™) (Life Technologies, Carlsbad, CA, USA) utilize an amplicon-based technology. Multiple primer pairs are employed to select target gene regions by PCR, which requires as little as 10 ng (or even less) DNA input and only 1–3 h to generate the sequence results [23]. Another NGS platform, the GeneReader NGS System(™) (Qiagen, Hilden, Germany), more recently became available. This platform requires at least 40 ng of DNA, adopts a hybridization-based library preparation methodology, and requires a relatively long analysis time (approximately 30 h) [27].

Clonal amplification is the second step in the NGS workflow. To enhance the chemical signal in the subsequent sequencing reaction, each single fragment of the library needs to be clonally expanded in hundreds of thousands of copies [15]. On the Illumina platform, clonal amplification takes place on a solid support of a flat glass microfluidic channel (flow cell) by the so-called bridge amplification [22], whereas the Ion Torrent and GeneReader platforms carry out clonal amplification by emulsion PCR on beads [5, 21, 27].

The third step in the NGS workflow is the massive parallel sequencing with generation of hundreds of thousands to millions of reads in parallel for each run [14, 27]. The differences among the most commonly adopted platforms are highlighted in Table 5.3. Despite the differences in DNA input requirements, the run times, read lengths, and costs per sample, the two most popular bench-top sequencing platforms (Illumina and Ion Torrent) produce comparable results [16].

Table 5.3 Difference between the commonly used NGS platforms in clinical laboratories
Full size table

Finally, sequencing data are analyzed by using a combination of software pipelines (Fig. 5.3) [27, 28]. This process requires four major steps: base calling, read alignment, variant identification, and variant annotation [29, 30]. The combination of informatics tools used for processing, aligning, and detecting variants in NGS data is commonly referred to as the bioinformatics pipeline. This process requires careful optimization at the time of validation to ensure that a variant call is effectively present in the sequence as well as continued quality control, as bioinformatics is constantly evolving. The necessity of validation of NGS technologies prior to clinical implementation cannot be overemphasized. Validation includes the identification of positive percentage agreement (PPA) and positive predictive value (PPV), the reproducibility of variant detection, the determination of the reference range, limits of detection (LOD), clinical and analytical sensitivity and specificity, and if appropriate, the validation of bioinformatics pipelines, and other parameters [31].

Figure 5.3
figure 3

EGFR mutation analysis by NGS. Read alignment visualization of Golden Helix GenomeBrowse v.2.0.7 (Bozeman, MT, USA) software showing an epidermal growth factor receptor (EGFR) exon 19 deletion (p.E746_A750delELREA)

Full size image

NGS is a powerful and versatile technique. A variety of gene panels is commercially available and can be classified in four distinct groups, as summarized in Table 5.4 [31, 32].

Table 5.4 Examples of gene panels
Full size table

The versatility of NGS lies in its ability to use custom panels to improve analytical performance and laboratory cost-effectiveness [3, 33]. Although it is widely held that NGS is an expensive technique, our experience with the commercially available AmpliSeq Colon and Lung Cancer Panel, which covers 22 genes involved in colon and lung cancer, showed that the consumable cost is only €196 ($238) per sample [25]. Moreover, the cost per sample could be even reduced to €98 ($119) by the use of a narrow gene panel targeting 568 clinically relevant mutations in 6 genes (EGFR, KRAS, NRAS, BRAF, KIT, and PDGFRA) [33].

In summary, NGS-based assays on routine cytology samples have the potential to improve patient care through diagnostic, prognostic, and predictive biomarker assessment. The basic principles of NGS described in this chapter underscore the need of a new generation of molecular cytopathologists [34,35,36].