Sample types applied for molecular diagnosis of therapeutic management of advanced non-small cell lung cancer in the precision medicine
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Yanxi Han
Abstract
In this era of precision medicine, molecular biology is becoming increasingly significant for the diagnosis and therapeutic management of non-small cell lung cancer. The specimen as the primary element of the whole testing flow is particularly important for maintaining the accuracy of gene alteration testing. Presently, the main sample types applied in routine diagnosis are tissue and cytology biopsies. Liquid biopsies are considered as the most promising alternatives when tissue and cytology samples are not available. Each sample type possesses its own strengths and weaknesses, pertaining to the disparity of sampling, preparation and preservation procedures, the heterogeneity of inter- or intratumors, the tumor cellularity (percentage and number of tumor cells) of specimens, etc., and none of them can individually be a “one size to fit all”. Therefore, in this review, we summarized the strengths and weaknesses of different sample types that are widely used in clinical practice, offered solutions to reduce the negative impact of the samples and proposed an optimized strategy for choice of samples during the entire diagnostic course. We hope to provide valuable information to laboratories for choosing optimal clinical specimens to achieve comprehensive functional genomic landscapes and formulate individually tailored treatment plans for NSCLC patients that are in advanced stages.
Introduction
Precision medicine classifies patients with the same disease further into finer subtypes based on their molecular biomarkers and offers them tailored therapies, with the hope of ensuring optimal benefits and concomitantly minimizing the risks, undesirable side effects and needless medical care [1], [2]. The advancement of this concept has revolutionized the diagnosis and therapeutic management of diseases, especially made great strides in the field of oncology. Here, we take one of the “success stories” of precision medicine-advanced non-small cell lung cancer (NSCLC) as a case study.
Lung cancers have the highest incidence of all cancers and are the leading cause of cancer-related deaths worldwide, with about 1.8 million new cases and 1.6 million cancer-related deaths per year [3], [4]. Only 17% of people diagnosed with lung cancers exhibit 5-year survivals [5]. Traditionally, lung cancers were classified per their histology into NSCLC, and small cell lung cancer (SCLC) [6]. NSCLCs account for 80%–90% of newly diagnosed cases of lung cancers. However, the pathology of lung cancers is complex, and distinct genetic differences in patients cause different rates of responses to the same treatments, even if they share similar histological characteristics [2]. In 2004, the seminal discovery of activating epidermal growth factor receptor (EGFR) mutations that confer sensitivity to tyrosine kinase inhibitors (TKIs) in adenocarcinomas of NSCLCs heralded the beginning of the era of precision medicine that was based on the molecular diagnoses of NSCLCs [7], [8]. In the following studies, researchers have demonstrated that besides EGFR mutations, anaplastic lymphoma kinase (ALK) rearrangements, Kirsten rat sarcoma viral oncogene (KRAS) mutations, human epidermal growth factor receptor-2 mutations and amplifications, v-Raf murine sarcoma viral oncogene homolog B mutations, rat c-ros oncogene 1 rearrangements, transfection oncogene rearrangements, encoding the proto-oncogene tyrosine kinase c-mesenchymal epithelial transition factor amplifications and exon 14 skipping serve as therapeutic targets [9], [10]. According to the latest National Comprehensive Cancer Network (NCCN) guidelines (Version 2.2017), EGFR mutations and ALK rearrangements should be evaluated routinely (category 1) in patients with advanced nonsquamous NSCLCs and in patients with squamous NSCLCs that exhibit atypical clinical features (e.g. squamous cell carcinomas in non-smokers, or small biopsies or mixed histology). ROS-1 rearrangements first appear explicitly as sole therapeutic targets, in the NCCN guidelines. The College of American Pathologists (CAP)/International Association for the Study of Lung Cancer (IASLC)/Association for Molecular Pathology Guideline (AMP) guidelines suggest that molecular testing should be completed at the time of confirmed diagnosis of advanced disease or at the time of recurrence [11]. Additionally, they encourage patients with early stage disease (stages I–III) who undergo tissue biopsies to additionally undergo molecular testing, to provide information to oncologists at the time of cancer recurrence [11].
Molecular testing plays a key role in the diagnostic and therapeutic stratifications of lung cancers, especially of the advanced NSCLCs, and has become a standard of clinical practice. As the primary elements of the whole testing flow, satisfactory specimens that allow for histological and molecular analysis are particularly important for ensuring accuracy of testing and preventing false-negative results and test failures.
Surgical resections were the first to be applied to clinical molecular diagnosis. Because of their large dimensions that provided sufficient tumor cells to enable testing by various methodologies and a clear assessment of tumor cellularity via hematoxylin-eosin (HE) staining, surgical resections had been the gold standard of sample types for molecular diagnosis [12]. However, about 50%–70% of patients with NSCLCs are advanced at diagnosis and are unresectable [13]. The use of newer and less invasive sampling techniques, such as bronchoscopy, thoracoscopy, CT-guided core biopsy, fine needle aspiration (FNA), etc., has shown promising results in collection of good quality and quantity of tumor cells, thus rendering small and cytology biopsies the most available materials for gene alteration testing [14]. According to statistics, the diagnosis and molecular profiling of 25%–30% NSCLCs are made by small biopsies, and up to 40% by cytology biopsies [15]. The success rates of both small and cytology biopsies range between 83% and 100% in published studies [16], [17], [18], [19], [20], [21]. It is known that 90% of patients with early stage NSCLCs who receive surgical resections or have locally advanced NSCLCs and receive definitive chemoradiation, eventually relapse [22]. After about a year of remission, most advanced lung cancers treated with TKIs acquire resistance, hence demonstrating that the main mutations develop earlier and the subclonal mutations later [23], [24]. Therefore, it is necessary to conduct repeat biopsies during the entire course of progression of NSCLCs, which would provide critical evidence for developing further treatment strategies. However, biopsies are highly invasive and may increase the risk and probability of tumor metastasis. It is unfeasible to use these biopsies to obtain tumor samples from patients exhibiting drug resistance or relapses after treatments. Liquid biopsy emerges as a noninvasive method that overcomes the issues of tumor heterogeneity and brings great hopes for the potential early detection and posttreatment monitoring of lung cancers [25], [26]. Liquid biopsy was rated as one of the top 10 breakthrough technologies of 2015 by the Massachusetts Institute of Technology Technology Review. The American Society of Clinical Oncology indicates that liquid biopsy is one of the most influential techniques for cancer field over the next decade [27].
The sample types we mentioned above vary in terms of sampling procedures and preparation and have their individual strengths and weaknesses. For now, there is no consensus on which sample type is preferable for accurate clinical management of lung cancers. Therefore, this review aims to summarize the strengths and weaknesses of different sample types that are widely used in clinical practice for diagnosis and monitoring of NSCLCs, to offer solutions to potentially overcome the flaws of the samples used in such cases and to propose an optimized strategy for choosing samples at each stage during the diagnostic course of advanced NSCLCs.
Characteristics of the samples available for molecular diagnosis in clinical practice
Tissue biopsy
Tissue biopsy was earliest applied to molecular diagnosis and was considered the gold standard for it. Depending on the techniques utilized for sampling, tissue biopsies range from large surgical resections to small biopsies obtained by endoscopy or by image-guided transthoracic core-needle biopsy [28].
Tissue biopsies provide relatively larger sampling materials. However, they also abundantly possess non-tumor-specific components, which may dilute tumor DNA and inhibit detections. It is reported that tissue biopsies, especially the small biopsies, are deeply influenced by tumor cellularity [29]. Therefore, the pathological evaluations of tissue biopsies and demarcations of ideal sampling regions are integral for identification of available tumor tissues and to guarantee the provision of sufficient DNA (>500 ng) for downstream testing [30]. The CAP/IASLC/AMP guidelines recommended that for EGFR gene mutations testing, 50% tumor cells are strongly encouraged, which is rarely achieved in routine practice; when using sensitive detection methods, tumor cells as little as 10% are acceptable [11]. Currently, as the technologies with high sensitivity (1%–0.01%), such as amplification refractory mutation system (ARMS), digital PCRs, and next-generation sequencing (NGS), are applied for gene alterations testing, the percentage of tumor cells as low as 1% can be reported [20]. For testing ALK rearrangements, the percentages of tumor cells used are not as critical as those for testing of EGFR mutations, but it is important to choose areas where tumor cells are not overlapping with one another and can be readily distinguished [12]. For poor quality specimens, a manual macro- or microdissection enrichment strategy, which can result in highly purified tumor cells for DNA extractions, or repeated biopsies, are requisite [15]. Enrichment strategies, such as laser capture microdissection or flow cytometric sorting for isolation of tumor cells from small biopsies, should be used cautiously because of their typically low yields of DNA.
Generally, tissue biopsies are prepared into fresh-frozen samples and formalin-fixed paraffin-embedded (FFPE) samples. Fresh-frozen tissues are routinely reviewed by pathologists as intraoperative diagnostic procedures to guide the surgery. They are embedded in an optimum cutting temperature compound, frozen rapidly at −80 °C or immersed in liquid nitrogen (−190 °C), then cut into serial frozen sections at −15 °C to −25 °C, using a cryostat for histological and molecular diagnosis [31]. Compared with FFPE samples, frozen tissues are optimal for providing high-quality DNA or RNA and are more conducive to analysis of long DNA segments (>1000 base pairs or bp) [32]. ALK rearrangement detections based on reverse transcription-polymerase chain reaction (RT-PCR) are allowed to be performed on frozen samples [33]. Frozen samples have shorter sample processing and staining time and have lower risks of producing DNA artifacts that are usually caused by formalin fixation. One drawback is that the fast sample processing and staining may lead to ambiguity in cell morphology. Sometimes, it may be quite difficult to identify exactly the site of tumor from normal tissues, and it is truly needed by experienced pathologists to process and read the frozen slides. The other drawback is that the freezing process may result in freezer burns at the periphery of samples; this may cause changes in the molecular profiles of the tissues. Thus, frozen samples are used only for distinguishing between benign and malignant tumors during operation rather than for histological classifications [34]. To lessen the damaging effect of ice crystals, the surgical samples of lung tissues containing vacuum structures could be immersed in 20%–30% sucrose solution to reduce their water content using the hypertonic principle. Once freezing temperature is ascertained, the freezing process should be completed as soon as possible to avoid the increased tissue fragility resulting from overfreezing and the uneven tissue sections resulting from underfreezing [35]. Another drawback pertains to the highly controlled storage conditions: fresh surgical samples need to be kept in liquid nitrogen, and the sections that are mounted on glass slides are required to be kept at −80 °C, until further use.
Although fresh tissues are ideal for conducting molecular tests due to the minimal processing required and the high quality of the resultant DNA, the overwhelming majority of laboratories still prefer FFPE samples. This is because FFPE samples provide commendable cellular morphology and preserve the entire tumor structure for assessment of tumor cellularity and diagnoses of lung cancers, which is vastly superior to the advantages of high-quality fresh-frozen samples [12]. Additionally, FFPE samples can be kept in archives at room temperatures for years [36], [37]; these constitute documentation for patients with NSCLCs and rich resources for clinical retrospective studies. However, the speed of formalin fixation is slow, at around 1 mm/h, and the DNA fragmentation, chemical cross-linking and artificial mutations caused by formalin fixation are well-recognized limitations [34]. The DNA fragmentation and cross-linking render DNA extraction and purification difficult to achieve and inhibit the length-dependent DNA amplification reactions to some extent. It is reported that testing reactions that require DNA segments (from FFPE samples) that are shorter than 300 bp are usually successful; those requiring DNA segments between 300 and 1000 bp succeed inconsistently, and those requiring longer than 1000 bp often fail [34]. Therefore, in lieu of RT-PCR, fluorescence in situ hybridization (FISH) is recommended as the gold standard for detection of ALK rearrangements in routine FFPE samples [33]. Additionally, formalin fixation generates random sequence artifacts [38], which can potentially obfuscate the gene alteration testing, typically in samples with low concentrations of DNA templates or those used in ultrasensitive assays [39]. The most frequently encountered type of sequence artifact is the single-base change of the transitional C:G>T:A variants resulting from the incorporation of an adenine opposite to the thymine or uracil residues by the DNA polymerase enzyme [38]; this type of sequence artifact happens to be consistent with EGFR T790M (c.2639T>C) point mutation. This underlines the need for caution in distinguishing sequence artifacts from the naturally occurring T790M mutation. To minimize the adverse effects of formalin fixation in FFPE samples, the following measures should be adopted: (1) preanalysis of the tumor cell ratios to ensure adequate amounts of amplifiable DNA templates and performance of manual enrichments in cases with abundant nonspecific tumor contents; (2) fixation of samples in cold 10% neutral formalin (at 4 °C) in the shortest possible times, typically 8–18 h for large surgical resections, and 6–12 h for small biopsies [28]; (3) use of heat treatment during DNA extraction, usually at >90 °C, to remove formalin-induced cross-links and to facilitate tissue digestion with proteinase K [40]; (4) pretreatment of DNA extracted by uracil-DNA glycosylase in order to prevent modified bases [41]; (5) use of short amplicons and application of unique molecular tagging combined with bidirectional strand sequencing of DNAs to distinguish sequence artifacts from naturally occurring mutations [42]; (6) performance of amplifications of FFPE samples in duplicates to ensure accurate results [12]; and (7) utilization of specific DNA polymerases that do not read through uracil residues and abasic sites and use of high-fidelity DNA polymerases to decrease polymerase-induced errors, when using PCR-based methods [38].
In addition, the sampling of tissue biopsies from primary tumors or metastases is just a snapshot in time and site and is not representative of the dynamics of the intact tumors [43]. Additionally, the presence of highly invasive tumors renders this approach more challenging to for repeat biopsies. Therefore, tissue biopsies are not ideal for monitoring tumor progressions and resistance during treatments.
Cytology biopsy
As mention above, cytology biopsies can be appropriate alternatives to tissue biopsies for accurate diagnoses. Cytology biopsies are obtained via less invasive procedures, including exfoliated cells collected from sputum or pleural effusion, biopsies obtained by endobronchial ultrasound-guided fine needle aspiration (EBUS-FNA), endoscopic ultrasonography-guided fine needle aspiration (EUS-FNA) and computed tomography-guided FNA, bronchoalveolar washing and bronchial brushing [20]. These are easier to be obtained from patients with advanced NSCLCs who experience poor health conditions. Researches on using sputum for noninvasive detections of early stage lung cancers have showed a promising future for routine diagnoses and potential for screening [44].
The use of less invasive procedures results in smaller amount of content in cytology samples, which may cause false-negative results [28]. To overcome the limitations of small sample sizes, cytological rapid on-site evaluations (C-ROSE) should be applied during sampling to assess the adequateness of the biopsies [45]. The lesion sites containing more tumor cells or the histologic cores are the best options for collection of more tumor-specific materials, and these are less affected by tumor cellularity. As reported, the performance of cytology biopsies was comparable to that of surgical resections, and they produced better results than those obtained by small biopsies [14], [21], [46]. When highly sensitive detection methods, such as ARMS PCR, peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp or NGS are applied, the success rates of cytology biopsies are pretty high [46], [47], [48]. Cytology samples containing more than 100–200 cells can successfully be detected by methods such as Sanger sequencing [49], [50]. Liu et al. and Sun et al. who utilized the ARMS PCR and the pyrosequencing method, respectively, reported that cytology specimens that satisfied any of the following three parameters, i.e. DNA concentrations of >2–25 ng/μL, tumor cells >30, or a tumor percentage of >25%–30%, showed a 100% concordance with the corresponding tissue biopsies [51], [52]. Among the cytology samples, pleural effusion has relatively higher rates of occurrence of EGFR mutations because most malignant tumor cells in pleural effusions are derived from advanced NSCLCs [53]. Nonetheless, when using cytology samples to detect T790M mutation, which is a secondary and low frequency mutation of the EGFR gene [23], specimens with as few as 5% of tumor cells and detection assays with higher sensitivities should be prioritized, and negative results should be interpreted with caution [11]. Guidelines recommend obtaining the use of maximal samples to overcome tumor heterogeneity and guarantee the accuracy of testing [54], but this needs to be balanced with the limited number of biopsies realistically possible, and the risks, such as pneumothorax or hemoptysis, involved in procedural complications [55]. To judiciously utilize precious cytology samples, appropriate protocols for histochemical and molecular analyses should be established. For instance, during preparation of cell blocks, multiple unstained sections for both histological and molecular analyses should be prepared when the sample is first processed to avoid wastage caused by refacing, and the substantial number of unstained sections lost during the refacing of cell blocks could be used for molecular testing [12].
Generally, cytology samples are prepared into smears (air-dry and 95% ethanol-fixed smears) and cell blocks (95% ethanol-fixed and FFPE cell blocks), the latter being the most widely applicable source of cells [15]. Air-dry smears are stained with Diff-Quik (DQ) or Giemsa, ethanol-fixed smears with Papanicolaou (PAP), and the cell block sections with HE. All slides need to be evaluated by pathologists; the tumor-rich areas are scrapped and utilized for DNA extractions and molecular testing. The air-dry smears stained by DQ are usually used for C-ROSE [56]. Both types of smears and the ethanol-fixed cell blocks can offer high-quality DNA, without the chemical cross-linking and the risks of DNA fragmentations found in FFPE samples [32], and are suitable for long fragment analysis. Furthermore, the processing time for cytology samples is much shorter than those for tissue samples. Cell blocks can be archived stably for conducting additional ancillary tests, when necessary [57], [58]. In some laboratories, smears and cell blocks are made simultaneously, usually histologic cores are used to prepare cell blocks, while the remaining material is smeared onto slides for smears [56]. It is reported that ethanol fixation may alter the morphological details of the cellular nuclei; therefore, it is more challenging to perform tests associated with morphology, especially FISH, on ethanol-fixed sections [12], [28]. Thus, some laboratories prefer to use cytology samples from FFPE cell blocks.
Moreover, as they can be obtained repeatedly and less invasively, cytology biopsies, especially the exfoliated cells collected from sputum or pleural effusion, are more suitable for monitoring the progressions, relapses and resistance of tumors and the histological transitions during treatments.
Liquid biopsy
Lung cancers harbor substantial genetic heterogeneity and evolve over time [59], [60], [61], [62]. Biopsies based on single site-specific sampling are insufficient for performing definitive diagnoses and therapeutic management. Liquid biopsies, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), can provide comprehensive information on the genetic landscapes of tumors, to overcome tumor heterogeneity and to dynamically monitor tumors [63], [64]. This shows promise as a minimally invasive, convenient and cost-effective method of molecular diagnosis [28].
Circulating tumor cells
Circulating tumor cells (CTCs) detach from primary or metastatic tumors, enter blood circulation and eventually form new metastases [65]. They have tumor-specific antigenic and genetic characteristics, which are intermediate between those of primary tumors and disseminated metastases. In patients with solid tumors, the presence of CTCs is the evidence for the presence of advanced stages of disease. Previously, the increased numbers of CTCs in cancers have been utilized for the predictions of prognoses and overall survivals in patients [66], [67].
However, the identification and isolation of CTCs present the main technical challenges to their clinical utility [68]. This is because CTCs are strongly influenced by tumor burden encountered substantial apoptosis and necroptosis during circulation and are rarely released in the peripheral blood [63]. Reportedly, there is only one CTC in 106–107 white blood cells (WBCs) per mL of blood [69]. Krebs et al. [67] reported that the CTCs in 7.5 mL of blood in patients with stage IV NSCLCs (n=60; range, 0–146) were more than those observed in patients with stage IIIB (n=27; range, 0–3) and IIIA (n=14; no CTCs detected) NSCLCs. For enrichment of CTCs, methods based on their physical (e.g. size, density and electric charge) or biological (e.g. surface protein expression and invasive capacity) properties have been developed [70]. The only system approved by the United States Food and Drug Administration (US FDA) for enrichment and enumeration of CTCs is the CellSearch® system, which enriches isolated CTCs by using magnetic bead-conjugated antibodies against epithelial-cell adhesion molecules (EpCAM) in 7.5 mL of blood [70]. However, a large body of evidence has demonstrated that EpCAMs is not universal makers for isolating CTCs, especially for those CTCs that have undergone epithelial-mesenchymal transition or have originated from the mesenchymal [70]. There is an urgent need to improve existing technologies or develop new systems for enrichment or isolation of CTCs.
Although there are many successful examples of CTC-based detections of gene mutations [71], [72], the use of CTCs usually shows no advantages over that of ctDNA because of the tumor heterogeneity and the miniscule amounts of DNA provided by the CTCs that are isolated by the existing platforms. However, CTCs highlight their advantages when they serve as biomarkers for immunotherapy, especially those that express PDL-1 [73]. With the preponderance of minimally invasive sampling procedures, CTCs are one of the most sensitive indicators for real-time monitoring of the progressions and relapses of lung cancers and the effectiveness of their treatments. The dynamics of CTCs are in accordance with the computed tomography (CT) responses by the response evaluation criteria in solid tumors criteria and the metabolic response determined by FDG-positron emission tomography (FDG-PET) [74]. Meanwhile, CTCs may help to fill the gray areas of radiography when tumor enlargement is <20% or tumor reduction is ≦30% or both and may assist in discovering early tumor disseminations; they can be the perfect complements to radiographic evidence. Single-cell molecular analysis based on CTCs can be applied as a research tool to reveal the mechanisms of metastases and resistance to targeted therapy [75], [76]. Additionally, CTCs could be useful for the following purposes: as noninvasive surrogates for HE staining to definitively diagnose NSCLCs, for FISH testing to guide ALK-targeted therapy and for mRT-PCR testing to detect the MET and HER3 gene amplifications that are related to the resistance to EGFR-TKI therapy, when suitable tissue biopsies are unavailable [77], [78].
Although CTCs are valuable genetic and phenotypic biomarkers, the current numbers and isolation methods of CTCs limit their broad applications in clinical practice, especially for patients with early stage NSCLCs.
Circulating tumor DNA
Circulating cell-free DNA (ccfDNA) was initially described by Mandel and Metais in 1948 [79]. Those that were shed from apoptosis, necrosis or secretions of tumor cells were named as circulating tumor DNA (ctDNA) [80]. By 1994, ctDNA was first utilized for detections of RAS gene mutations [81]. In the recent decades, the potential roles of ctDNA as tumor biomarkers are becoming increasingly valuable. Presently, ctDNA have been used in clinical practice as one of the sample types for gene alteration testing. In 2015, the US FDA approved Therascreen® EGFR RGQ PCR kit (QIAgen) as the liquid biopsy-based companion diagnostic test for IRESSA (generic name: gefitinib). Further, in 2016, the Cobas EGFR Mutation Test v2 (Roche) for Tarceva (generic name: erlotinib) has been approved.
CtDNAs are relatively stable blood- or urine-borne DNA segments [26], [63], [82]. The origins of ctDNAs determine their homogeneity and tumor specificity, as well as their ability to represent the entire tumor landscape and provide reliable evidence for identification of genetic determinations for targeted therapy [83]. It is reported that the analysis of ctDNA has showed mutations that have been missed in the matching tissue samples [84], [85], [86]. CtDNAs can be obtained serially via minimally invasive sampling procedures, and their half-lives are comparatively shorter (<2 h) [87]. The serial analysis of driver mutations in ctDNAs could demonstrate the response to targeted therapy and monitor the evolution of multiple resistant clones, which impact the ongoing treatment decision making and patient survival. A case report on the abundance of the EGFR T790M mutant during the treatment of NSCLCs with the third-generation EGFR-TKI, AZD9291 (generic name: osimertinib), demonstrated rapid increases in levels of mutant ctDNAs (with the exon 19 deletions and exon 20 T790M mutations) 3 days after treatment with AZD9291, indicating probable tumor lysis. This was followed by rapid decreases in levels of both these mutations the next day; these decreases were probably associated with the responses to treatments, and the mutations were barely detectable 6 days after treatment [88]. Piotrowska et al. performed longitudinal ctDNA analysis to reveal the different resistant mechanisms of rociletinib, a third-generation EGFR-TKI [89]. Among 12 T790M-positive NSCLC patients with rociletinib treatment, six had T790M-wild-type rociletinib-resistant clone, two underwent small cell lung cancer (SCLC) transformation and three acquired EGFR-activating mutations, which suppressed the T790M clone. To improve outcomes, other targeted agents or combination regimens need to be applied. These findings demonstrated the key role of ctDNA identifying the multiple resistant clones during the treatment course. Changes in ctDNA can proceed weeks to months before those observed in imaging studies or in studies using protein biomarkers [25], [90]. Therefore, ctDNAs are ideal samples for early detection of tumor progression and relapse [25], [91], [92], [93], [94]. Oxnard et al. used the serial plasma genotyping of EGFR mutations in lung cancers to demonstrate the dynamics of EGFR mutations from pretreatments to complete plasma responses in most cases, as well as the increasing levels of the EGFR T790M mutant emerging prior to tumor progression, as observed by imaging studies [95].
Additionally, ctDNA made the absolute quantification of driver mutations feasible. Unlike tissue and cytology samples, which show different gene alteration copy numbers corresponding to different tumor loci, ctDNA is the optimal and at present probably the only available medium for absolute quantitative detection of these mutations. In 2011, Zhou et al. [96] demonstrated that the quantification of EGFR mutations may enable further predictions of benefits from EGFR-TKI therapy. Patients with a higher abundance of EGFR mutations may experience increased benefits with EGFR-TKI therapy. Because of the limitations of tissue samples and detection methods, the research team derived no further specific conclusions. Subsequently, in 2016, Zhou et al. [97] demonstrated that patients with different dynamic types of plasma L858R mutations benefited differently from EGFR-TKI therapy. The median progression-free survivals of patients with the ascended type of plasma L858R mutations (L858R increased to its highest level during disease progression) and the stable type (L858R maintaining stable levels during disease progression) were 11.1 (95% CI, 6.6–15.6) and 7.5 months (95% CI, 1.4–13.6), respectively, and the overall survivals (OS) were 19.7 (95% CI, 16.5–22.9) and 16.0 months (95% CI, 13.4–18.5), respectively [97]. Martin et al. [98] similarly concluded that the percentages of EGFR mutations in NSCLCs have close associations with the responses to EGFR-TKI therapy, the time to treatment failure (TTF) and the OS. It is supposed that gene amplifications either facilitated the development of gene mutations or amplified their effects [99]. In the recent 3 years, with the increasing research on ctDNA and digital PCR, the precise measurement of the abundance of driver mutations needs to be as significant as their detection. This paradigm shift is the more intensive and rigorous interpretation of precision medicine. Till date, large-scale clinical trials are in progress, and there is no clarity on an effective threshold of the abundance of driver mutations for TKI targeted therapy, and more attention needs to be focused on the quantification of gene alterations.
Much like gene alterations in ctDNA, methylation changes also can be detected in circulation and correspond to tumor burden [25]. DNA methylation is often an early event in tumor, which makes it a potential tool for early screening of tumor [100]. DNA methylation and nucleosome occupancy patterns in ctDNA have been confirmed to be tumor specific and cell specific [101], thus helping to aid tumor localization [102].
However, effective detection of ctDNA remains challenging. The concentration of total circulating cell-free DNA (ccf-DNA) ranges from 0 to 1000 ng/mL (the average is 180 ng/μL) in cancer patients. CtDNA comprises a very small fraction of ccf-DNA (<1%) [87], [103], and the DNA with gene alterations accounts for only 0.02%–0.1% of total ccf-DNA [104]. This means that the presence of mutant alleles will be diluted in the background of wild-type genomic DNA (gDNA) and introduce biological noise [102]. Nonetheless, combined with highly sensitive detection methods, such as ARMS PCR (0.1%–1%) [105], [106], NGS (0.1%–0.02%) [107], [108], digital PCR (0.01%) [95], [109] or beads emulsion magnetic cytometry (BEAMing) (0.01%) [87], [110], the specificity and sensitivity of liquid biopsies is 95%–100% and 62%–93%, respectively. The rate of concordance between matched tissues and liquid biopsies is 72%–91%, and higher concordance rates can be observed in patients with stage III and IV disease [106], [111], [112], [113]. The close relationship between ctDNA abundance and tumor stage limits the applications of ctDNA in early stage patients (stage I–IIIA). Uchida et al. conducted EGFR mutation testing by deep sequencing (n=288). The findings showed that the sensitivity in all cases was 54.4% (95% CI 44.8%–63.7%); in stages IA–IIIA, 22.2% (11.5%–38.3%); and in stages IIIB–IV, 72.7% (60.9%–82.1%) [114]. Ct-DNA is highly fragmented, ranging from 160 to 200 bp [115]. The most abundant fragments are approximately 180 bp long [116]; this raises difficulties in designing testing assays and renders these fragments unsuitable for long fragment analysis.
To obtain reliable results, the preanalysis of ctDNA should be highly prioritized and standardized. The optimal source of ctDNA is plasma, rather than serum, and the required volume ranges from 1.5 to 5 mL [117], depending on the different DNA extractions and detection assays used. The processing of ctDNA is simple but demanding. First the blood samples need to be collected in EDTA anticoagulant tubes [118]; cell-free DNA™ blood collection tubes are preferred for their ability to stably preserve blood samples containing ctDNA for 14 days at room temperature [119]. Second, considering that ctDNA is highly fragmented, blood samples need to be processed into plasma within 30–40 min after blood drawing, and no more than 6 h after [117]. Third, the genomic DNA (gDNA) released by necrotic WBCs may dilute the ctDNA fraction in plasma, rendering ctDNA detection more challenging [80]. Thus, double centrifugation (1200–1600 g for 10 min followed by 16,000 g for 10 min) should be performed to reduce contamination with gDNA [117]. Fourth, it is recommended that the extraction of ctDNA be performed with commercial ctDNA extraction kits with high yield, like the QIAamp DNA Circulating Nucleic Acid Kit (Qiagen) and Applied Biosystems™ MagMAX™ Cell-Free DNA Isolation Kit (ThermoFish) [120]. Finally, plasma samples should be aliquoted for further gene alteration testing or storage at −80 °C for long-term preservation [121].
Optimized choosing strategy of sample type
The median untreated life expectancy of patients with stage IV lung cancers is approximately 16 weeks [12]. The completion of histological and molecular diagnosis requires about 2 weeks (i.e. 10 working days) [12], [54], which means more than 10% of the untreated life expectancy time is spent on waiting for the testing results. As time is critical in the care of patients with advanced NSCLCs, we want to provide a stepwise strategy for choice of samples based on the strengths and weaknesses of each sample type, to facilitate faster completion of testing and initiation of reasonable therapy. Given that patients with advanced NSCLCs experience poor health conditions, the least invasive biopsy with the highest yield is preferred, in the whole diagnostic course. Figure 1 shows the sequence of events in histological and molecular diagnoses for lung cancers and the optimized sample types we recommended at each step.
Flow chart of histology and molecular diagnosis for advanced NSCLCs and the optimized sampled types recommended for each step.
First, identify lung cancers by imaging studies. Second, establish a definitive diagnosis, including histological classification and stage, by tissue or cytology biopsies. Third, determine the gene alterations associated with the targeted therapy by using tissue or cytology biopsies. Liquid biopsies are the appropriate alternatives for patients with advanced disease whose tissue and cytology biopsies are not available. Finally, monitor the tumor progressions, resistance to targeted therapy and histological transitions during treatments, using liquid or exfoliative cytology biopsies or a combination of both. *Patients with nonsquamous NSCLCs, and patients with squamous NSCLCs who exhibit atypical clinical features (e.g. squamous cell carcinomas in a nonsmoker, or a small biopsy or a mixed histology). **Patients with locally advanced (stage IV) and early stage (stages I–III) NSCLCs with recurrences or distant metastases or a combination of both. ***When the testing for gene alterations performed on liquid biopsy shows negative results, other confirmatory tests based on tissue or cytology biopsies obtained at the appropriate times should be performed.
Following a provisional diagnosis by imaging studies, the suspicious lesions require a tissue-based diagnosis to definitively determine histological classification and stage. Cytology specimens are the acceptable alternatives when tissue biopsies cannot be obtained. Immediately after a histological diagnosis is established, the molecular testing associated with the identification of mutated oncogene therapeutic targets should be initiated. Currently, no consensus has been reached regarding the difference in the status of gene alterations between primary and metastatic lesions. According to CAP/IASLC/AMP guidelines, the primary tumor and its metastases are equally suitable for the detection of gene alterations [11]. For patients with advanced or metastatic NSCLCs whose tissue and cytology biopsies are unavailable or inadequate for molecular testing, ctDNA and CTCs are the preferred sample types for screening driver mutations and gene rearrangements, respectively. This is because the collection procedures for ctDNA or CTCs are minimally invasive, which circumvents the potential for injury associated with invasive sampling procedures; and the processing of such samples is simple, which shortens turnaround time greatly, and facilitates rapid clinical decisions. However, a caveat of these procedures is that if negative results are obtained for all targeted genes with clear significance, other confirmatory tests need to be performed to exclude the possibility of false-negative results. For assessment of the tumor dynamics, exfoliative cytology biopsies and liquid biopsies are more competent. The serial quantitative detection of ctDNA and CTCs can enable the early evaluation of tumor prognosis and response to targeted therapy. Because ctDNA samples are much less prone to heterogeneity, compared with biopsies and CTCs, the continuous assessment of ctDNA is extremely useful for discovering resistant alterations during targeted therapy and in assisting the development of further treatment strategies. Besides, the monitoring of CTCs, sputum or pleural effusion by exfoliative cytology over time may unravel the histological transition from NSCLCs to SCLCs. The characteristics of commonly used methods for detecting molecular alterations in NSCLC specimens were summarized in Table 1. What need laboratories to arise attention is that assays with high sensitivity are at risk of yielding false-positive results. Therefore, the sensitivity and specificity of a chosen assay should be balanced, and the clinical threshold of mutant allele to effective target agents needs to be established in order to ensure that targeted therapies are in appropriate management.
Characteristics of methods for detecting gene alterations in NSCLC specimens.
| Method | Sensitivity (%mutant DNA) | Mutations identified | Optimal application of sample type | Reference |
|---|---|---|---|---|
| Sanger sequencing | 20–30 | Known and new | Tissue and cytology biopsy | [25], [122] |
| PCR-SSCP | 10 | Known and new | Tissue and cytology biopsy | [123] |
| Pyrosequencing | 5–10 | Known only | Tissue and cytology biopsy | [25] |
| HRMA | 2.5–10 | Known and new | Tissue and cytology biopsy | [124], [125] |
| MALDI-TOF MS-based genotyping | 5 | Known only | Tissue and cytology biopsy | [126] |
| TaqMan PCR | 1–5 | Known only | Tissue and cytology biopsy | [25] |
| dHPLC | 1 | Known and new | Tissue and cytology biopsy | [127], [128] |
| PNA-LNA PCR clamp | 0.1–1 | Known only | Tissue and cytology biopsy, ctDNA | [47], [84] |
| ARMS PCR | 0.1–1 | Known only | Tissue and cytology biopsy, ctDNA | [25], [105] |
| Mutant-enriched PCR | 0.2 | Known only | Tissue and cytology biopsy, ctDNA | [129], [130] |
| NGS-based deep sequencing | 0.1–0.02 | Known and new | ctDNA, rare variants in tissue and cytology biopsy | [86], [100], [107] |
| Digital PCR | 0.01 or lower | Known only | ctDNA, rare variants in tissue and cytology biopsy | [25], [102], [131] |
| BEAMing | 0.01 or lower | Known only | ctDNA, rare variants in tissue and cytology biopsy | [25], [103], [132] |
PCR-SSCP, polymerase chain reaction-single-strand conformation polymorphism; HRMA, high-resolution melting analysis; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; dHPLC, denaturing high-performance liquid chromatography; PNA-LNA, peptide nucleic acid – locked nucleic acid; ARMS, amplified refractory mutation system; BEAMing, beads, emulsions, amplification and magnetics.
Conclusions and prospects
In the era of precision medicine, the identified actionable gene alterations have been used to subdivide NSCLCs into more nuanced subtypes and guide clinical decision making, and the scope of this process is steadily increasing. Selection of satisfactory samples is the first and the paramount step for accurate molecular genotyping. As discussed in this review, there are strengths and weaknesses associated with each sample type (Table 2), with respect to different parameters, such as sampling and preparation procedure (Figure 2), the heterogeneity of inter- or intratumors, the quality and percentage of tumor-specific components, etc., and no single sample type can individually be a “one size to fit all”. In practice, each laboratory should establish the minimal sample requirements (i.e. proportion and number of tumor cells) for the detection platform during validation [11], [28]. Pathologists need to be involved in the preanalytic step for the assessment of tumor architecture and cellularity for molecular analysis, to choose reasonably adequate specimens, and to minimize the risk of false-negative results [133], [134]. Once collected, the tumor samples need to offer high-quality molecular data to identify truly predictive gene biomarkers correlating with the clinical diagnosis and treatment of NSCLCs.
Strengths and weaknesses of different sample types.
| Sample type | Advantages | Disadvantages | Reference | ||
|---|---|---|---|---|---|
| Tissue sample | |||||
| (i) Fresh-frozen sample | – | Shorten the sample processing and staining time | – | Be obtained via invasive sampling procedure | [12], [15], [28], [31], [32], [33], [34], [35], [42], [54] |
| – | Provide relatively large sample dimension | – | Rapid freezing result in freezer burn and may change the molecular profile of the tissue | ||
| – | Preserve entire tissue structure and good antigenic activity | – | Affected by tumor cellularity significantly | ||
| – | Offer high-quality DNA or RNA with low risk of fragmentation and artifacts | – | Require highly controlled conditions for processing, cutting and storing | ||
| – | Can be preserved long in −80 °C or in liquid nitrogen (−190 °C) | – | Unsuited for monitoring the tumor progress, relapse and resistance during treatment for it’s just a snapshot in time and site | ||
| – | Can be applied to most gene alteration detection methods | ||||
| – | Be suitable for long fragment analysis | ||||
| – | Can be used for intraoperative diagnosis to guide the surgery and for analysis of both histologic and molecular diagnosis | ||||
| (ii) FFPE tissue sample | – | Provide relatively large sample dimension | – | Be obtained via invasive sampling procedure | [12], [14], [15], [28], [29], [33], [34], [36], [37], [38], [39], [40], [41], [42] |
| – | Providing commendable cellular morphology and preserve entire tissue structure | – | Require long time for fixation (6–18 h) | ||
| – | Can be kept in the archives at room temperature | – | Affected by tumor cellularity significantly | ||
| – | Can be applied to most gene alteration detection methods | – | Undergo cumbersome process of DNA extraction | ||
| – | Can be used for histologic and molecular diagnosis and for clinical retrospective studies | – | Offer the DNA with fragmentation, chemical cross-linking and sequence artifacts caused by formalin fixation | ||
| – | Unsuitable for length-dependent amplification reaction and monitoring tumor the progress, relapse and resistance during treatment | ||||
| Cytology biopsy | |||||
| (i) Smear | – | Be obtained via less invasive sampling procedure and allowed repeated biopsy | – | Contain small amount of overall cell content and may cause false-negative results | [28], [30], [32], [34], [45], [46], [49], [55], [56] |
| – | Shorten the sample processing time greatly | – | Require high sensitive detection methods | ||
| – | Provide higher quality of DNA with low risk of fragmentation or artifacts | – | May cause cell shrinkage, alter the morphological details of the nucleus and not suitable for FISH testing (95% ethanol fixed smear) | ||
| – | Can be used for C-ROSE during sampling | ||||
| – | Provide more tumor-specific materials and be less affected by tumor cellularity | ||||
| – | Be suitable for patients whose tissue biopsies are not available | ||||
| – | Be suitable for long fragment analysis | ||||
| – | Can be applied to monitor the progress and relapse of tumor and resistance during treatment | ||||
| (ii) Cell block | – | Be obtained via less invasive sampling procedure and allowed repeated biopsy | – | Contain small amount of overall cell content and may cause false-negative results | [12], [15], [16], [28], [38], [44], [46], [49], [53], [55], [56], [57], [58] |
| – | Provide more tumor-specific materials and be less affected by tumor cellularity | – | Require high sensitive detection methods | ||
| – | Provide higher quality of DNA with low risk of fragmentation or artifacts (95% ethanol fixed cell block) | – | May cause cell shrinkage, alter the morphological details of the nucleus and not suitable for FISH testing (95% ethanol fixed cell block) | ||
| – | Be suitable for patients whose tissue biopsies are not available | – | Require long time (6–18 h) for fixation (formalin fixed cell block) | ||
| – | Can be archived at room temperature stably for additional ancillary tests | – | Undergo cumbersome process of DNA extraction (formalin fixed cell block) | ||
| – | Can be used for analysis of both histology and gene alteration analysis and for clinical retrospective studies | – | The DNA fragmentation, cross-linking and sequence artifacts caused by formalin fixation (formalin fixed cell block) | ||
| – | Can be applied to monitor the progress and relapse of tumor and resistance during treatment | – | Unsuitable for length-dependent amplification reaction (formalin fixed cell block) | ||
| Liquid biopsy | |||||
| (i) CTCs | – | Be obtained via minimally invasive sampling procedure and allowed repeated biopsy | – | The amount of CTCs is very small result in difficulties to identify and isolate | [63], [66], [67], [68], [69], [70], [71], [73], [74], [75], [76], [77], [78] |
| – | Be more tumor-specific | – | Need large volume of blood | ||
| – | Can be used as surrogates for HE staining of tumor diagnosis | – | Be affected significantly by tumor burden | ||
| – | Can be applied to FISH for gene rearrangement detection and to mRT-PCR for gene amplification detection | – | Require extremely sensitive and specific detection methods | ||
| – | Serve as the sensitive predictor for monitoring treatment effectiveness, prognosis and overall survival of tumor | – | Confine its application to advanced patients | ||
| – | Can be applied as a research tool to reveal the mechanisms of metastasis and resistance | – | Still in research level | ||
| (ii) CtDNA | – | Exist stably in blood or urine as fresh DNA segment | – | The overall amount of ctDNA is very small portion (<1%) of ccfDNA and may cause false negative results | [25], [26], [63], [80], [82], [83], [84], [85], [87], [89], [91], [92], [93], [97], [107], [108], [109], [110], [114] |
| – | Represent the landscape of whole tumor better for its homogeneity and tumor-specific | – | Require more sophisticated DNA extraction methods | ||
| – | Be obtained via minimally invasive sampling procedure and allowed repeated biopsy | – | Be affected significantly by tumor burden | ||
| – | The sample procedure is very simple and time-saving | – | Require extremely sensitive and specific methods | ||
| – | Be with short half-life time (<2 h) | – | Be highly fragmented and unsuitable for long fragment analysis | ||
| – | Serve as the sensitive predictor for tracing tumor dynamics, early detection of the minimal residual lesions and recurrence and monitoring the response and resistance of targeted therapy | – | Require highly controlled preservation conditions (–80 °C) due to its degradation at room temperature | ||
| – | Make absolute quantification of gene alterations feasible | – | Confine its application to advanced patients | ||
Sample preparation procedures.
For tissue biopsies and cell blocks, serial sections should be cut, and HE-stained sections need to be analyzed by experienced pathologists to formulate definitive histologic diagnoses and evaluate the tumor cellularity. The tumor areas from 2 to 10 sections with the least amount of necrosis, blood, mucous or inflammation should be submitted for DNA extractions and molecular testing. For cytology smears, C-ROSE should be performed during sampling to evaluate the adequateness of the biopsies. The regions rich in tumor cells should be scraped for DNA extraction and molecular testing. For liquid biopsies, 1.5–5 mL plasma and CTCs that are enriched and isolated by physical or biological approaches can be collected for DNA extractions and molecular testing.
Although open surgical biopsies are generally preferred, there are many factors that affect the decisions regarding putative samples used for histological and molecular diagnosis, and for further ancillary testing [14]. For patients with stage IV NSCLCs, tissue biopsies are usually not available. With the trend toward minimally invasive and safe sampling procedures, liquid biopsies (involving ctDNA and CTCs) become the ideal alternatives. Liquid biopsies demonstrate promising prospects for monitoring of responses and resistance during treatments, early detection of prognoses and recurrences of tumors and, absolute quantitative detections of gene alterations [96], [97]. Currently, the use of ctDNA for ancillary testing has been approved by the FDA in Europe and China. However, studies showed that the amounts of ctDNA and CTCs are associated closely with cancer stage and blood-flow resistance (e.g. blood-brain barrier) in patients [111]. Research on ctDNA and CTCs for use in the diagnosis of patients with early stage disease is still ongoing [135]. Liquid biopsy is the appropriate alternative for patients with advanced disease whose tissue biopsies cannot be obtained and can be used as an initial screening tool for early diagnosis, alongside with the tissue-based approaches [111]. Tissue-based molecular analysis remains the accepted standard for establishing the initial diagnosis to facilitate decisions regarding clinical treatments, as well as, for monitoring resistance to targeted therapies [136].
The successful treatments of patients with NSCLCs who harbored EGFR mutants with EGFR-TKIs therapy prompted researchers to investigate other novel therapeutic targets [137]. Advances in genomic technology, such as NGS, have expanded the breadth and depth of the detection of gene alterations and have made it possible to analyze broad molecular profiling simultaneously, for discovering the comprehensive functional therapeutic targets in lung cancers [138], [139], [140]. The implementation of NGS permits the output of sequences with megabases to gigabases of genetic information in a single run using a limited amount of input DNA; this has enabled a shift in detection from single genes towards multigene panels [141]. Using such gene panels with multiple potential targets will definitely increase the likelihood of ensuring appropriate treatment for patients with lung cancers [142]. In addition, the sensitivity of NGS in the detection of gene alterations has significantly increased to 0.02%–1% [108], [143], allowing the detection of rare somatic alterations or variations in heterogeneous tumors [141]. Currently, the expansion of numbers of these gene alterations seems to pose some problems. One of these problems that need to be surmounted is the necessarily longer time required to interpret data on whole gene sequences [144]. Another problem pertains to generating a readable report of the alterations that are identified while complying with the best practice guidelines [145], especially for the novel alterations in already known and putative targets that can guide therapy [146]. Therefore, the laboratories applying NGS for clinical gene alteration testing must exert massive efforts toward the interpretation of testing results and reinforce the expertise and consultations required to support the platform.
The molecular testing in precision medicine is still at an exploratory stage. In every diagnostic laboratory, precise workflow and optimized standard operating procedures should be established for appropriately selecting and processing samples because these are the factors that determine successful molecular diagnosis. Technologies for isolation and detection should be verified or validated with respect to their accuracy, sensitivity, specificity and repeatability and cross-validated by experienced lab personnel before being applied in clinical practice [76]. Additionally, there is an increased need to form seamless multidisciplinary cooperative consortiums to provide individualized diagnostic and therapeutic care plans for patients with advanced lung cancers.
Author contributions: Yanxi Han wrote the whole manuscript; Jinming Li gave the most valuable advice to this work. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This work was supported by a grant from the Special Fund for Health Scientific Research in the Public Interest from National Population and Family Planning Commission of the People’s Republic China (No. 201402018). It was not supported by any private or public company or organization.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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©2017 Walter de Gruyter GmbH, Berlin/Boston
Articles in the same Issue
- Frontmatter
- Editorial
- Tumor microenvironment and systemic disease: a dual target in medical oncology (also in the case of biomarkers)
- Reviews
- Sample types applied for molecular diagnosis of therapeutic management of advanced non-small cell lung cancer in the precision medicine
- The impact of pneumatic tube system on routine laboratory parameters: a systematic review and meta-analysis
- Opinion Paper
- How to reduce scientific irreproducibility: the 5-year reflection
- EFLM Paper
- Strategies to define performance specifications in laboratory medicine: 3 years on from the Milan Strategic Conference
- General Clinical Chemistry and Laboratory Medicine
- Intensive educational efforts combined with external quality assessment improve the preanalytical phase in general practitioner offices and nursing homes
- Separate patient serum sodium medians from males and females provide independent information on analytical bias
- Are admission procalcitonin levels universal mortality predictors across different medical emergency patient populations? Results from the multi-national, prospective, observational TRIAGE study
- Biochemical testing in a laboratory tent and semi-intensive care of Ebola patients on-site in a remote part of Guinea: a paradigm shift based on a bleach-sensitive point-of-care device
- Efficient reporting of the estimated glomerular filtration rate without height in pediatric patients with cancer
- Evaluation of thyroid test utilization through analysis of population-level data
- Relation of serum γ-glutamyl transferase activity with copper in an adult population
- Impact of a single oral dose of 100,000 IU vitamin D3 on profiles of serum 25(OH)D3 and its metabolites 24,25(OH)2D3, 3-epi-25(OH)D3, and 1,25(OH)2D3 in adults with vitamin D insufficiency
- Automated antinuclear immunofluorescence antibody analysis is a reliable approach in routine clinical laboratories
- Infrared analyzers for breast milk analysis: fat levels can influence the accuracy of protein measurements
- Hematology and Coagulation
- A new approach to define acceptance limits for hematology in external quality assessment schemes
- The effects of transport by car on coagulation tests
- Combined measurement of factor XIII and D-dimer is helpful for differential diagnosis in patients with suspected pulmonary embolism
- Reference Values and Biological Variations
- Influence of age, gender and body mass index on late-night salivary cortisol in healthy adults
- Cancer Diagnostics
- Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients
- Detection of EGFR mutations in patients with non-small cell lung cancer by high resolution melting. Comparison with other methods
- Predicting outcomes of EGFR-targeted therapy in non-small cell lung cancer patients using pleural effusions samples and peptide nucleic acid probe assay
- Analytical and clinical performance of thyroglobulin autoantibody assays in thyroid cancer follow-up
- Thyroglobulin autoantibodies before radioiodine ablation predict differentiated thyroid cancer outcome
- Cardiovascular Diseases
- Real life dabigatran and metabolite concentrations, focused on inter-patient variability and assay differences in patients with atrial fibrillation
- Infectious Diseases
- HIV avidity index performance using a modified fourth-generation immunoassay to detect recent HIV infections
- Letters to the Editor
- Effect of age on serum prostate-specific antigen in women
- Falsely elevated thyroid-stimulating hormone value due to anti-ruthenium antibodies in a patient with primary hypothyroidism: a case report
- High titers of anti-infliximab antibody do not interfere with Abbott immunoassays
- Extraordinarily elevated serum CA19-9 in a patient with posterior mediastinum cyst: a case report
- Low levels of 25-OH vitamin D in women with endometriosis and associated pelvic pain
- Evaluation of serum cortisol biological variation in the evening withdrawal
- Distinction between urine crystals by automated urine analyzer SediMAX conTRUST is specific but lacks sensitivity
- Impact of heat-inactivation on anti-Toxoplasma IgM antibody levels
Articles in the same Issue
- Frontmatter
- Editorial
- Tumor microenvironment and systemic disease: a dual target in medical oncology (also in the case of biomarkers)
- Reviews
- Sample types applied for molecular diagnosis of therapeutic management of advanced non-small cell lung cancer in the precision medicine
- The impact of pneumatic tube system on routine laboratory parameters: a systematic review and meta-analysis
- Opinion Paper
- How to reduce scientific irreproducibility: the 5-year reflection
- EFLM Paper
- Strategies to define performance specifications in laboratory medicine: 3 years on from the Milan Strategic Conference
- General Clinical Chemistry and Laboratory Medicine
- Intensive educational efforts combined with external quality assessment improve the preanalytical phase in general practitioner offices and nursing homes
- Separate patient serum sodium medians from males and females provide independent information on analytical bias
- Are admission procalcitonin levels universal mortality predictors across different medical emergency patient populations? Results from the multi-national, prospective, observational TRIAGE study
- Biochemical testing in a laboratory tent and semi-intensive care of Ebola patients on-site in a remote part of Guinea: a paradigm shift based on a bleach-sensitive point-of-care device
- Efficient reporting of the estimated glomerular filtration rate without height in pediatric patients with cancer
- Evaluation of thyroid test utilization through analysis of population-level data
- Relation of serum γ-glutamyl transferase activity with copper in an adult population
- Impact of a single oral dose of 100,000 IU vitamin D3 on profiles of serum 25(OH)D3 and its metabolites 24,25(OH)2D3, 3-epi-25(OH)D3, and 1,25(OH)2D3 in adults with vitamin D insufficiency
- Automated antinuclear immunofluorescence antibody analysis is a reliable approach in routine clinical laboratories
- Infrared analyzers for breast milk analysis: fat levels can influence the accuracy of protein measurements
- Hematology and Coagulation
- A new approach to define acceptance limits for hematology in external quality assessment schemes
- The effects of transport by car on coagulation tests
- Combined measurement of factor XIII and D-dimer is helpful for differential diagnosis in patients with suspected pulmonary embolism
- Reference Values and Biological Variations
- Influence of age, gender and body mass index on late-night salivary cortisol in healthy adults
- Cancer Diagnostics
- Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients
- Detection of EGFR mutations in patients with non-small cell lung cancer by high resolution melting. Comparison with other methods
- Predicting outcomes of EGFR-targeted therapy in non-small cell lung cancer patients using pleural effusions samples and peptide nucleic acid probe assay
- Analytical and clinical performance of thyroglobulin autoantibody assays in thyroid cancer follow-up
- Thyroglobulin autoantibodies before radioiodine ablation predict differentiated thyroid cancer outcome
- Cardiovascular Diseases
- Real life dabigatran and metabolite concentrations, focused on inter-patient variability and assay differences in patients with atrial fibrillation
- Infectious Diseases
- HIV avidity index performance using a modified fourth-generation immunoassay to detect recent HIV infections
- Letters to the Editor
- Effect of age on serum prostate-specific antigen in women
- Falsely elevated thyroid-stimulating hormone value due to anti-ruthenium antibodies in a patient with primary hypothyroidism: a case report
- High titers of anti-infliximab antibody do not interfere with Abbott immunoassays
- Extraordinarily elevated serum CA19-9 in a patient with posterior mediastinum cyst: a case report
- Low levels of 25-OH vitamin D in women with endometriosis and associated pelvic pain
- Evaluation of serum cortisol biological variation in the evening withdrawal
- Distinction between urine crystals by automated urine analyzer SediMAX conTRUST is specific but lacks sensitivity
- Impact of heat-inactivation on anti-Toxoplasma IgM antibody levels
