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Pellerino et al. J Cancer Metastasis Treat 2020;6:41 I http://dx.doi.org/10.20517/2394-4722.2020.80 Page 7 of 20

including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), or exosomes. CSF liquid biopsy
has been suggested as a more sensitive tool to achieve a diagnosis of LM than conventional CSF cytology.
CTCs can be found using rare cell capture technology and immune flow cytometry assay with antibodies
against epithelial cell adhesion molecule (EpCAM), reporting a sensitivity of 76%-100% and a specificity
[62]
[63]
of 84%-100% for diagnosis of LM . van Bussel et al. reported a sensitivity of 94% (95%CI: 80-99) and
a specificity of 100% (95%CI: 91-100), with a cutoff of 0.9 CTC/mL, when using EpCAM immunoflow
cytometry. Several studies have shown that the CellSearch technique with immunomagnetic identification
and quantification of CSF CTCs improves the ability to diagnose LM from NSCLC, particularly in those
patients with conventional acquired negative CSF cytology [64-66] . Moreover, the addition of CSF ctDNA
analysis may improve the diagnosis of LM in cases with a low amount of CSF CTCs . Plasma liquid
[67]
biopsy has been investigated as a surrogate tool for diagnosis of LM from NSCLC. Unfortunately, poor
concordance has been reported between plasma and CSF in LM from NSCLC regardless of the type of
[68]
driver mutation and liquid biopsy technique. Zheng et al. have reported that next-generation sequencing
of paired plasma and CSF samples of 11 patients with LM from ALK rearranged NSCLC identified driver
[69]
mutations in 81.8% of CSF and 45.5% only of plasma. Similarly, Ying et al. have compared CSF and
plasma samples of 92 patients with LM from EGFR mutated NSCLC reporting a high mutation rate in CSF
(81.5%) with an overall amount of 197 mutations, whereas plasma displayed a lower mutation rate (62.5%)
and amount of mutations (68%). Furthermore, a significant discordance of mutation profiles between
CSF and plasma has been reported: a further analysis of EGFR showed an activating mutation in 51.4%
of CSF and 38.9% of plasma samples with a concordance of 47.7%. Notably, the EGFR T790M resistance
mutation was detected in CSF of 2 patients only (2.8%), denoting that mutation occurs more frequently in
extracranial sites [67,70,71] . Huang et al. reported similar results when evaluating EGFR status in CSF and
[72]
plasma (75% vs. 36.4%, respectively) in a cohort of 11 LM cases from EGFR mutated NSCLC, and EGFR
[73]
T790M mutation was found more frequently in plasma (39%) compared with CSF (13%). Li et al. have
found targetable EGFR mutations in CSF of 26/26 patients (100%) and in plasma of 19/26 patients (73.1%).
In particular, TP53 loss of heterozygosity (LOH) was identified in CSF of 19/26 patients (73.1%) and in
plasma of 2/26 patients only (7.7%), and T790M mutation in 8/26 CSF samples (30.4%) and in 6/26 plasma
[74]
samples (21.7%). Lastly, Ma et al. reported that 7/13 patients (53.8%), who received TKIs, developed
uncommon EGFR mutations in ctDNA of CSF. Interestingly, these uncommon EGFR mutations, including
G719A, L861Q, L703P, and G575R mutations, were more frequent in LM (54.5%) than in patients with
BM (10%). Overall, CSF liquid biopsy appears to be more sensitive than plasma in detecting druggable
mutations in LM. Moreover, CSF has a significant number of specific mutations, such as TP53 LOH,
MET amplification, CDKN2A, NTRK1 and CDK4 mutations, that contribute to the tumorigenesis and
development of LM from NSCLC [69,75] .

Integrated assessment for diagnosis and monitoring of LM from NSCLC
The combination of CSF analysis, including cytology and liquid biopsy, with MRI assessment may improve
[58]
the ability to diagnose LM. Hyun et al. studied the diagnosis of LM in a cohort of 519 patients with
advanced NSCLC; by MRI alone in 35% of patients, by CSF cytology alone in 22%, and by both techniques
[4]
in 42% . However, the absence of a standardized quantification of LM disease burden represents a
[76]
challenge. Recently, Nevel et al. have investigated whether MRI disease burden assessment and CSF
analysis can be employed to predict survival in LM from NSCLC. For the MRI assessment, the Authors
have scored the MRI using 8 predefined anatomic locations, such as cerebrum, ventricles, brainstem,
cerebellum, cranial nerves, cervical, thoracic, and lumbosacral spinal cord. One point has been awarded
for each affected location regardless of the number of lesions. The presence of ≥ 3 sites of disease was
statistically correlated with a risk of death compared with < 3 sites (HR = 1.95; 95%CI: 1.16-3.30; P = 0.01),
while anatomic locations of LM were not associated with OS. Regarding CSF analysis, protein level above
the upper limit was not correlated with OS, while CSF white blood cell count and low glucose at diagnosis
[76]
of LM were significantly correlated with OS (P = 0.04 and P < 0.0001, respectively). Importantly, Nevel et al.
reported that patients with ≥ 50 CTCs/3 mL had an increased risk of death in comparison with those with <