Diagnosis and pathology/molecular biology

Diagnosis

Changes in the therapeutic scenario in the last 15 years have emphasised the need for a multidisciplinary approach in lung cancer. Data show that high-volume centres and multidisciplinary teams are more efficient at managing patients with lung cancer than low-volume or non-multidisciplinary centres, by providing more complete staging, better adherence to guidelines and increased survival [ 21, 22 ]. Multidisciplinary tumour boards influence providers’ initial plans in 26%–40% of cases [ 23 ]. The absolute need to reach a proper and precise morphological and biological definition often requires challenging tissue sampling, with most treatment decisions depending on the information obtained from the specimen collected at diagnosis.

Bronchoscopy is a technique ideally suited to large, central lesions and offers the advantage of minimal morbidity. Bronchoscopy can be used for bronchial washing, brushing, bronchial and transbronchial biopsy, with a diagnostic yield of 65%–88% [ 24-26 ]. By combining direct bronchoscopic airway visualisation with ultrasound-guided biopsy of the lesion, endobronchial ultrasound (EBUS) provides a diagnostic yield of 75%–85% in large, centrally located lesions [ 27,28 ]. Fibre optic bronchoscopy allows for the evaluation of regional lymph nodes by EBUS and/or endoscopic ultrasound (EUS). EBUS-guided transbronchial needle aspiration (TBNA) is less invasive and at least as accurate as mediastinoscopy[ 29 ]. Several studies have shown that cytological specimens obtained by EBUSTBNA are suitable for molecular testing for epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homologue (KRAS) and anaplastic lymphoma kinase (ALK) status [ 30-33 ]; however, collection of samples suitable for broader molecular diagnostic testing should be encouraged.

In case of peripheral lesions, transthoracic percutaneous fine needle aspiration and/or core biopsy, under imaging guidance [typically computed tomography (CT)] is proposed [ 34 ]. Needle biopsy is associated with a diagnostic accuracy of > 88% yield, a sensitivity of 90% and a false-negative rate of 22% [ 25,35-38 ]. The most significant disadvantage of transthoracic needle biopsy is a procedural risk of pneumothorax, ranging from 17% to 50% [ 37,38 ].

In the presence of a pleural effusion, thoracentesis could represent both a diagnostic tool and a palliative treatment. If fluid cytology examination is negative, image-guided pleural biopsy or surgical thoracoscopy should be carried out. More invasive, surgical approaches [mediastinoscopy, mediastinotomy, thoracoscopy, video-assisted thorascopic surgery (VATS), secondary lesion resection etc.] in the diagnostic workup are considered when the previously described techniques cannot allow for an accurate diagnosis.

Pathology/molecular biology

Histological diagnosis

Histological diagnosis of NSCLC is crucial to many treatment decisions and should be as exact and detailed as the samples and available technology allow. Diagnosis should be based upon the criteria laid out in the WHO classification [ 39 ]. This classification details the complete diagnostic approach for surgically resected tumours but, importantly, also provides guidance for assessing and reporting small biopsy and cytology samples where complete morphological criteria for specific diagnosis may not be met [ 39-41 ].

Most patients with NSCLC present with advanced stage unresectable disease, therefore all treatment-determining diagnoses must be made on small biopsy and/or cytology-type samples. Sampling may be carried out of the primary tumour or any accessible metastases, taken under direct vision or more usually with image-guided assistance, which greatly increases the diagnostic yield (hit rate). Sampling metastatic disease may facilitate staging, as well as diagnosis. These diagnostic samples frequently have limited tumour material and must therefore be handled accordingly; ensuring processing is suitable for all likely diagnostic procedures and that material is used sparingly at each step, since many diagnostic tests may be required [ 42 ].

Immunohistochemistry (IHC) has become a key technique in primary diagnosis as well as in predictive biomarker assessment. In those cases of NSCLC where specific subtyping is not possible by morphology alone, a limited panel of IHC is recommended to determine the subtype [ 39,40 ]. Thyroid transcription factor 1 (TTF1) positivity is associated with probable diagnosis of adenocarcinoma, p40 positivity with probable diagnosis of SCC; if neither are positive the diagnosis remains NSCLC-not otherwise specified (NOS). IHC staining should be used to reduce the NSCLC-NOS rate to < 10% of cases diagnosed [IV, A]. Pathologists are urged to conserve tissue at every stage of diagnosis, to use only two tissue sections for IHCNSCLC subtyping and to avoid excessive IHC investigation, which may not be clinically relevant.

Molecular diagnostics

After morphological diagnosis, the next consideration is therapy-predictive biomarker testing. This practice will be driven by the availability of treatments and will vary widely between different geopolitical health systems [ 43-45 ]. Contemporary practice has now evolved into two testing streams, one for the detection of targetable, usually addictive, oncogenic alterations and the other for immuno-oncology therapy biomarker testing. A personalised medicine synopsis table is shown in Table 1.

Last updated: 18 September 2019
Several molecular drivers for oncogene addiction represent strong predictive biomarkers and excellent therapeutic targets. They are generally mutually exclusive of each other [ 43-45 ]. These tumours are much more common in never- (never smoked or who smoked < 100 cigarettes in lifetime), long-time ex- (>10 years) or light-smokers (<15 pack-years) but they can also be found in patients who smoke. The vast majority of oncogene-addicted lung cancers are adenocarcinomas. Patients, in general, tend to be younger, while female gender and East Asian ethnicity particularly enriches for EGFR-mutant tumours. Nonetheless, guidelines suggest that all patients with advanced, possible, probable or definite, adenocarcinoma should be tested for oncogenic drivers [ 43-46 ]. Molecular testing is not recommended in SCC, except in those rare circumstances when SCC is found in a never-, long-time ex- or light-smoker (<15 pack-years) [IV, A]. Testing for EGFR mutations and rearrangements involving the ALK and ROS1 genes are now considered mandatory in most European countries. BRAF V600E mutations are rapidly approaching this status as first-line BRAF/MEK inhibitors are more widely approved, while HER2 (human epidermal growth factor receptor 2) and MET exon 14 mutations and fusion genes involving RET and NTRK1 (neurotrophic tyrosine receptor kinase 1) are evolving targets/biomarkers [ 43-46 ].

EGFR tyrosine kinase inhibitors (TKIs) are established effective therapies in patients who have activating and sensitising mutations in exons 18–21 of EGFR [ 47 ]. Prevalence is around 10%–20% of a Caucasian population with adenocarcinoma but much higher in Asian population. Around 90% of the most common mutations comprise deletions in exon 19 and the L858R substitution mutation in exon 21. Any testing approach must cover these mutations [I, A]; however, complete coverage to include exons 18–21 is recommended [III, B]. The T790M exon 20 substitution mutation is only rarely found in EGFR TKI-naive disease using standard techniques but is the most frequent cause of resistance to first- and second-generation EGFR TKIs (50%–60% of cases). Cases of patients carrying germline T790M mutation have also been reported [ 48 ]. Further studies to better understand the prevalence, familial penetrance and lifetime lung cancer risk in germline T790M-mutant patients are warranted. Implications of this mutation in TKI-naive disease are unclear, but the availability of TKIs effective against T790M-mutant recurrent disease makes T790M testing on disease relapse mandatory [I, A]. Cell-free DNA (cfDNA) blood testing is an acceptable approach to detect T790M at relapse but lacks sensitivity, so all patients with a negative blood test still require tissue biopsy [II, A] [ 49 ]. Tissue biopsy may also be more effective in identifying other resistance mechanisms which may require alternative treatment (SCLC transformation, MET amplification, HER2 alterations etc.).

Table 1: A personalised medicine synopsis table for metastatic NSCLC

Biomarker Method Use LoE, GoR
EGFR mutation Any appropriate, validated method, subject to external quality assurance To select those patients with EGFR-sensitising mutations most likely to respond to EGFR TKI therapy I, A
ALK rearrangement Any appropriate, validated method, subject to external quality assurance. FISH is the historical standard but IHC is now becoming the primary therapy-determining test, provided the method is validated against FISH or some other orthogonal test approach. NGS is an emerging technology To select those patients with ALK gene rearrangements most likely to respond to ALK TKI therapy I, A
ROS1 rearrangement FISH is the trial-validated standard. IHC may be used to select patients for confirmatory FISH testing but currently lacks specificity. NGS is an emerging technology. External quality assurance is essential To select those patients with ROS1 gene rearrangements most likely to respond to ROS1 TKI therapy II, A
BRAF mutation Any appropriate, validated method, subject to external quality assurance To select those patients with BRAF V600-sensitising mutations most likely to respond to BRAF inhibitor, with or without MEK inhibitor therapy II, A
PD-L1 expression IHC to identify PD-L1 expression at the appropriate level and on the appropriate cell population(s) as determined by the intended drug and line of therapy. Only specific trial assays are validated. Internal and external quality assurance are essential To enrich for those patients more likely to benefit from anti-PD-1 or anti-PD-L1 therapy. For pembrolizumab, testing is a companion diagnostic for nivolumab and atezolizumab, testing is complementary I, A
ALK, anaplastic lymphoma kinase; EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridisation; GoR, grade of recommendation; IHC, immunohistochemistry; LoE, level of evidence; MEK, mitogen-activated protein kinase kinase; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; TKI, tyrosine kinase inhibitor.

Fusion genes involving ALK and a number of partners (most commonly EML4) account for around 2%–5%of the same population that is routinely tested for EGFR mutations [ 50 ]. ALK-driven adenocarcinoma is very sensitive to several ALK TKIs. Early trials validated break-apart fluorescent in situ hybridisation (FISH) as the test to identify ALK gene rearrangement but the close association between a positive FISH test and modestly elevated ALK protein in tumour cells allows ALK IHC to be used, either to select cases for confirmatory FISH testing or as the primary therapy-determining test [ 50,51 ]. ALK IHC must reliably detect low levels of ALK protein and be validated against alternative tests to detect ALK fusion genes, especially if ALK IHC is used as the therapy-determining assay, without confirmation by FISH [II, A]. Emerging data demonstrate that the presence of the ALK protein (positive IHC staining) is associated with treatment response [I, A] [ 52,53 ]. Recently, IHC has been accepted as an equivalent alternative to FISH for ALK testing [ 54 ]. Testing for ALK rearrangement should be systematically carried out in advanced non-squamous NSCLC [I, A]. ALK mutations are emerging as important resistance mechanisms to ALK TKIs and ALK mutation testing may soon become a routine test at relapse as newer-generation ALK TKIs show differential efficacy against different ALK mutations [ 55 ].

ROS1 fusion genes are yet another addictive oncogenic driver that occurs in ~1%–4% of the same testing population. Like ALKROS1 has several potential fusion gene partners. Crizotinib, a TKI effective against ALK and MET, is also approved by the European Medicines Agency (EMA) for use in ROS1-rearranged adenocarcinomas. FISH has been the standard approach to detecting ROS1 rearrangements. IHC may be used in a manner similar to ALK testing, to identify candidate tumours for confirmatory FISH testing. The sensitivity of this approach is high, using currently available IHC, but specificity of IHC is low [IV, C]. FISH or other testing is required to confirm the diagnosis; IHC is currently not recommended as the primary treatment determining test [IV, A] [ 45,46,50 ]. Testing for ROS1 rearrangement should be systematically carried out in advanced non-squamous NSCLC [III, A].

BRAF mutation testing is now required in many countries after the approval of BRAF and MEK inhibitors for BRAF V600-mutant NSCLC. Any method is valid provided that it is adequately sensitive for the samples used and has been appropriately quality-assured, both within the laboratory and through external quality assurance. The V600E mutation is the most common of the BRAF V600 family and, overall, these BRAF mutations are found in ~2% of cases. BRAF V600 mutations appear mutually exclusive to EGFR and KRAS mutations, ALK and ROS1 rearrangements and are similarly much more common in adenocarcinoma. BRAF V600 mutation status should be systematically analysed in advanced non-squamous NSCLC for the prescription of BRAF/MEK inhibitors [II, A].

For many laboratories, testing for EGFR and BRAF mutations and ALK and ROS1 rearrangements involves individual standalone tests. Multiplex, massively parallel, so-called next-generation sequencing (NGS) of various sorts is rapidly being adopted as the standard approach to screening adenocarcinomas for oncogenic targets [III, A] [ 45,49,50,56 ]. Platform-specific, commercially available panels can cover genes of interest and provide a comprehensive, multiplex test for mutations and, in some cases, fusion genes. NGS will not address biomarkers that require testing at the protein level (requires IHC) and the question of whether NGS-detected fusion genes require an orthogonal test (IHC, FISH) for confirmation remains open. Whatever testing modality is used, it is mandatory that adequate internal validation and quality control measures are in place and that laboratories participate in, and perform adequately, external quality assurance schemes for each biomarker test [III, A].

The approval of the anti-programmed cell death protein 1 (PD-1) agent pembrolizumab as a standard-of-care first-line treatment in selected patients has made programmed death-ligand (PD-L1) IHC a mandatory test in all patients with advanced NSCLC. Although the PD-L1 IHC 22C3 assay was the only test validated in clinical trials of pembrolizumab, extensive technical comparison studies suggest that trial-validated commercial kit assays based on the 28-8 and SP263 PD-L1 IHC clones may be alternative tests [III, A] [ 57-61 ]. If laboratories use, by choice or force of circumstances, a non-trial-validated PD-L1 IHC test, i.e. a laboratory developed test (LDT), there is a high risk that the assay may fail quality assurance and a very careful, extensive validation is essential before clinical use [IV, A] [ 35,36 ]. There is a relationship between the extent of PD-L1 expression on tumour cells, or in some trials in tumour infiltrating immune cells, and the probability of clinical benefit from numerous anti-PD-1 or PD-L1 agents, in first- and second-line therapy [ 57 ]. For pembrolizumab, the mandatory treatment threshold is a tumour proportion score (TPS, presence of PD-L1 signal on tumour cell membranes) ≥ 50% in first line and ≥ 1% in second line [ 62,63 ]. PD-L1 expression testing is recommended for all patients with newly diagnosed advanced NSCLC [I, A]. For nivolumab and atezolizumab in second line, PD-L1 testing is not required for drug prescription. PD-L1 IHC is an approved biomarker test for immunotherapeutics in NSCLC but it is not a perfect biomarker; less than half of biomarker-selected patients benefit from treatment and some responses may be encountered in ‘biomarker-negative’ cohorts. Much work is underway to identify alternative, or more likely, additional biomarkers to enrich patient populations for response. Various measures of tumour mutational burden (TMB) are being explored and TMB has been validated prospectively in a unique prospective clinical trial to date [ 64 ]. An international effort is ongoing to define a consensus on how TMB should be measured [ 65-67 ]. Assessment of tumour inflammation is also of interest, but again, various approaches are being pursued, including histological assessment of immune cell infiltrates and mRNA-based expression signatures of immune-related genes. More data are required before any of these new approaches can be routinely incorporated into NSCLC biomarker testing.

Blood monitoring

The ability to detect oncogenic driver genomic alterations, or factors associated with disease resistance to treatment in peripheral blood, opens the way to disease monitoring in a way that would not be practically feasible were repeat testing solely based upon tumour biopsy testing. In practice, and with current knowledge, this is more likely to involve the use of cfDNA rather than circulating tumour cells (CTCs); the vast majority of existing data concern EGFR mutation testing in blood [ 68 ]. Currently, much EGFR plasma testing is based upon highly sensitive allele-specific polymerase chain reaction (ASPCR). Plasma genotyping may be considered before undergoing a tumour biopsy to detect the T790M mutation. However, if the plasma testing is negative for T790M, the tissue biopsy is strongly recommended to determine T790M status because of the risks of false-negative plasma results [III, A]. NGS techniques can be used; as more biomarkers are identified and validated, more NGS-based gene panels would be available.

Notwithstanding the issues regarding sensitivity of blood testing, potentially clinically valuable information may be derived from serial blood testing during treatment. For example, the disappearance from the blood of the primary sensitising EGFR mutation is associated with clinical and radiological evidence of response to EGFR TKIs and is a good prognostic indicator [IV, C].

After maximum response to EGFR TKI therapy and disappearance of the mutation from the plasma, the reappearance of the primary sensitising mutation, with or without detection of the T790M resistance mutation, may be an indicator of ‘biochemical’ disease relapse. This occurrence may predate radiological relapse, which, in turn, may predate clinical/symptomatic disease relapse. Currently, such findings are essentially exploratory since there is no consensus as to when and how any clinical intervention should be managed. There is no doubt, however, that this kind of molecular monitoring could, in the future, offer benefit to patients in a number of different personalised treatment scenarios.

Last updated: 18 September 2019
TMB was evaluated in patient tissue as well as blood samples in different trials. Unique assays and cut-offs are not yet defined but preliminary data from the POPLAR and OAK trials found TMB in blood is associated with improved atezolizumab clinical benefit in patients with NSCLC [ 69 ]. Exploratory data suggesting blood TMB (bTMB) as a predictive biomarker for atezolizumab as well as durvalumab/tremelimumab activity front-line have recently been presented [ 70, 70a]. bTMB measured from ctDNA allows for rapid, less invasive testing and may be more representative of the heterogeneity of metastatic lesions. Two prospective trials in the first-line setting are exploring the same biomarker [NCT03178552; NCT02542293].

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