prehensive multiple biomarker testing in NSCLC, the analysis of distinct biological molecules (DNA, RNA, proteins) and the use of appropriate analytical platforms (PCR, DNA sequencing, immunohistochemistry, FISH) are required [86].

Liquid Biopsy

Liquid biopsy is performed by using minimally invasive technologies to detect circulating biomarkers (circulating tumor cells and nucleic acids including cell-free RNA, micro-RNA, and circulating cell-free DNA that includes cell-free circulating tumor DNA, exosomes, tumor-­associated antigens and tumor-educated platelets) in blood and other body fuids (pleural fuid, BAL fuid, saliva, cerebrospinal fuid, urine, etc.). Intact and often viable circulating tumor cells released into the bloodstream from the primary tumor or metastatic site can be used for DNA-, RNAand pro- tein-based analysis and may reveal the heterogeneity that cannot be shown by indirect molecular approaches. Despite advances in cell detection technologies, these fragile circulating tumor cells are quite rare. Thus, their detection rate in NSCLC is usually low. However, recently circulating tumor cells have been identi ed in pulmonary venous blood in 48% of resected lung cancers. This nding suggests that it can be a clinically useful test in the future with improvements in technology. Somatic mutations in primary tumors can be more easily detected in circulating tumor DNA than in circulating tumor cells. Highly sensitive blood-based assays can identify molecular alterations at very low concentrations of circulating tumor DNA by either a narrow approach using PCR to target short sequences of DNA or a broad approach using NGS to target broader regions of DNA and multiple genes [78, 87].

Although small non-coding RNA (including miRNA) is stabilized by processing circulatory proteins, cell-free RNA is degraded fast in the circulation. The miRNA can be used as a biomarker in diagnosis, screening and determining prognosis as it can be quanti ed by using quantitative reverse transcription-polymerase chain reaction. However, miRNA is not in clinical use yet because there are no standard set of markers and thresholds for positivity used in the related studies [78].

Improvements in the diagnostic performances of the assays have led to the entrance of liquid biopsies into routine clinical practice for non-­invasive genotyping and monitoring the disease course. NGS is increasingly used for cell-free DNA testing as it can sequence multiple targeted genomic regions simultaneously in shorter turnaround time and with reduced sample requirements [87].

Nonetheless, in advanced NSCLC tissue still remains as the issue for personalized medicine which depends on suf cient tissue for biomarker testing. Liquid biopsy is complementary to tissue biopsy in determining driver and resistance mutations but it cannot replace tissue biopsy currently. The major challenges in using liquid biopsy are lack of standardization in tests, low sensitivity in early lung cancer, posttreatment or in detecting minimal residual disease, and clonal hematopoiesis of uncertain clinical signi cance causing false positive results. In the right clinical context, liquid biopsy can be bene cial regarding risk strati cation, diagnosis, prognostication, monitoring, and decreasing the number of invasive procedures [78, 87].

Summary, Recommendations and Highlights

\1.\ Diagnosis and staging of lung cancer should be managed promptly and accurately by an ef cient process minimizing procedures before treatment.

\2.\ Within the multidisciplinary team approach to identify the best evidence-based treatment plan for lung cancer care, minimally invasive procedures provide rapid and safe acquisition of tissue for the diagnosis, staging, and molecular testing (Table 18.1).

\3.\ The possibility of the ideal tissue acquisition for simultaneous diagnosis, tumor classi cation, molecular testing and staging by the initial procedure depends on the individual patient and the need for suf cient and appropriate tissue for current and future cytological, immunohistochemical, and molecular studies.

\4.\ A systematic assessment of at least three mediastinal node stations including station 7 (subcarinal) is recommended as random or single-node sampling can be inadequate.

\5.\ A multimodality approach by combining diagnostic or staging techniques strategically provides more successful yields and better outcomes in the management, and may possibly be more cost-effective.

\6.\ For establishing a diagnosis of malignancy, subclassifying cancer reliably by using immunohistochemical stains, and for molecular analysis to determine targetable driver mutations, the obtained cytologic or histologic (small biopsy) specimens should be suf cient in quality and quantity. Thus, tissue with suf cient number of lung cancer cells is the issue.

\7.\ Whenever cytological samples are obtained, smears should be combined with cell block

preparations to increase the diagnostic yield and molecular adequacy.

\8.\ A panel of immunostains should be performed judiciously and in a focused manner to preserve cellular material for downstream molecular testing during the diagnostic work-up of a suspected NSCLC if histology or cytology by itself cannot distinguish squamous­ cell carcinoma from adenocarcinoma (Fig. 18.1).

\9.\ Molecular analysis of all lung adenocarcinomas (including mixed tumors having adenocarcinoma component) in advanced stage

may be performed for EGFR mutations by PCR-based techniques, and for ALK gene rearrangements by FISH assay or screening immunohistochemistry.

10\ .\ However, the increasing number of genomic targets for lung cancer and one-off testing approach in molecular analysis will result in

Fig. 18.1  A diagnostic algorithm for histologic subtyping and molecular analysis in treatment-­naive NSCLC patients [2, 4, 78, 82]. (*): next generation sequencing (NGS) preferred if available, CA cancer, NSCLC-NOS

non-small cell lung cancer histology not otherwise speci-ed, ctDNA cell-free circulating tumor DNA, SOC standard of care, PD-L1 programmed death ligand 1, IHC immunohistochemistry

the depletion of the cellular specimen although the cytopathologist can maximize cellularity of the cell block and minimize loss from the specimen in the initial work-up.

\11.\ Consequently, multiplexed panels for genomic analysis will be a must in the near future. Upfront NGS becomes the optimal and cost-effective strategy for an expanded panel beyond three biomarkers.

12\ .\ ROSE, sensitive genotyping assays (NGS) and/or liquid biopsy can be used to overcome challenges such as inadequate lung cancer tissue in the sample, histological and biological heterogeneity of the tumor, heterogeneous resistance mechanisms in the progressive tumor, and poor performance status of the patient.

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