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18F-FDG PET/CT total lesion glycolysis is associated with circulating tumor cell counts in patients with stage I to IIIA non-small cell lung cancer

Background In non-small cell lung cancer (NSCLC), 18F-fluoro-2-deoxy-D-glucose (18F-FDG) uptake determined by PET and presence of circulating tumor cells (CTCs) in the peripheral blood independently predict outcomes. For 18F-FDG PET/CT staging interpretation, standardized uptake values (SUVmax/avg) are routinely used in clinical reporting. The goal was to investigate whether 18F-FDG uptake measured by SUVmax/avg, but also measures of metabolic tumor volume (MTV) and total lesion glycolysis (TLG) (MTV × SUVavg), are associated with CTCs. Methods Prospectively, 7.5 mL blood was drawn from NSCLC patients at the time of staging 18F-FDG PET/CT and from healthy control subjects. CTCs were identified by immunofluorescent staining (CK8/18/19pos/EpCAMpos/CD45neg/DAPIpos nucleus). 18F-FDG PET/CTs were analyzed for SUVmax, SUVavg, MTV, and TLG. Results In 16 NSCLC patients with stage I–IIIA, MTV and TLG, in contrast to SUVmax and SUVavg, were positively associated with CTCs (linear regression analysis). No CTCs were detectable in 20 healthy control subjects. Conclusions This pilot study demonstrates that 18F-FDG PET/CT TLG correlates with CTCs in NSCLC patients without distant metastases. TLG might be a more appropriate marker for hematogenous micrometastatic potential than SUVs.


F-FDG uptake
Original Article 18 F-FDG PET/CT total lesion glycolysis is associated with circulating tumor cell counts in patients with stage I to IIIA non-small cell lung cancer parameters are predictive of poor outcome in NSCLC and other cancer types (3,4). Increased 18 F-FDG uptake on PET is associated with higher tumor glucose metabolism, which correlates with metastatic spread (5). Standardized uptake values (SUV avg/max ) are parameters typically reported in staging PET imaging for NSCLC, calculated as the ratio of radioactivity per unit volume of a region of interest (ROI) to the activity per unit whole body volume (6). However, combined measures of metabolic and volumetric tumor information, such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG) (calculated as MTV × SUV avg ), may be a more appropriate predictor for micrometastatic spread than SUVs (7,8).
Circulating tumor cells (CTCs)-defined as CK pos / EpCAM pos /CD45 neg with a DAPI pos nucleus-are found in the blood of cancer patients and correlate negatively with survival in NSCLC (9). CTCs have been implicated in development of metastases (10). Due to their simple detection, CTC liquid biomarker research is a most active field in translational oncology. The linkage between CTCs and tumor glucose metabolism is poorly understood (11). Since both CTCs and PET activity are associated with metastatic spread, we hypothesized that metabolic PET parameters correlate with CTCs in NSCLC. This is a pilot study that demonstrates that TLG is associated with CTCs in non-metastatic NSCLC patients, in contrast to SUV max/avg . Results suggest that TLG may be a better marker than SUVs for micrometastatic spread of NSCLC.

Subjects
Institutional Review Board approval was obtained (IRB2010166/IRB2004401-VA). Prospectively, treatmentnaïve NSCLC patients that underwent staging for surgery for stage I-IIIA (AJCC 8 th ed.) between July 2016 to December 2017 and healthy never-smokers were enrolled and signed informed consents.

F-FDG PET/CT
Imaging was performed with Biograph 32-Slice PET/CT scanners (Siemens, Germany), approximately one hour after 18 F-FDG intravenous injection using commercially available Medrad ® Intego PET Infusion System. Dose of 18 F-FDG injected was based on body weight and equaled 0.04 mCi/ pound body weight. The acquisition time per bed ranged from 1-4 minutes, based on BMI (with longest times applied for patients with severe morbid obesity). Patients fasted overnight and underwent blood glucose testing prior to scanning [median 95 mg/dL (range, 72-130)]. Attenuation correction of images was performed with low-dose CT without intravenous contrast. MIM Software (Cleveland, OH) was used to determine SUV max , SUV avg , MTV was calculated by 3D region isocontour-based VOIs (12). TLG was calculated as SUV avg × MTV.

CTC detection
Seven point five milliliter of blood were drawn in CellSave ® tubes (Menarini, Huntingdon Valley, PA), and CTCs were enriched by size with CellSieve ™ microfilter (Creatv MicroTech, Rockville, MD), as described (13,14). In alignment with the FDA-approved definition, CTCs were identified by immunofluorescence as CK pos /EpCAM pos / CD45 neg with a DAPI pos nucleus.

Statistical analysis
Research personnel were blinded to study-specific outcomes by separation from the recruiting staff. Statistical significance refers to a P<0.05. Analyses were performed using Prism 5.00 (GraphPad, La Jolla, CA).

Characteristics of subjects and CTC detection
In total, 36 subjects were prospectively recruited: 16 NSCLC patients and 20 healthy never-smokers ( Table 1). All NSCLC patients carried CTCs, however none of the 20 healthy controls had CTCs detectable ( Figure 1).

F-FDG-PET/CT uptake parameter associations with CTCs
To associate metabolic parameters with CTCs, linear regression analyses were performed ( Figure 2). We found a significant positive association between MTV and CTCs [slope= 0. 13    This pilot study results suggest that MTV and TLG, in contrast to SUV max/avg , are potential predictors for micrometastatic hematogenous spread measured by CTCs in NSCLC patients ( Figure 3).

Discussion
SUV max is the standard metabolic parameter applied for clinical NSCLC staging (15). However the TLG, a measure that takes the metabolic tumor volume burden into account, may provide a better reflection of cancer biology (7). Our findings suggest that in addition to MTV, TLG is a more accurate marker than SUVs for micrometastatic NSCLC spread represented by CTCs. Correlation of SUV max with poor outcome in NSCLC has been suggested, but conflicting data exist (3,5,16). Independently, 18 F-FDG PET/CT TLG and CTCs in the blood predict increased risk of recurrence in NSCLC, but the association of 18 F-FDG uptake parameters with CTCs is poorly understood (11,(17)(18)(19). We performed metabolic not in loco-regional lymph node metastases (if present). Although we did not include NSCLC patients with distant metastases, the study is limited by population heterogeneity. The majority of patients had lung tumors with no locoregional nodal metastases, but three patients had ipsilateral intrapulmonary/hilar and/or mediastinal lymph node metastases.
In metastatic NSCLC patients undergoing chemotherapy, a correlation between a decrease in CTCs and SUV max has been demonstrated (19). In another cohort of metastatic NSCLC patients that often have multiple tumor sites with different metabolic uptake, no correlation between CTCs and metabolic parameters, including TLG, was noted (20). In contrast, our study focused on non-metastatic NSCLC patients that might be less diverse than metastatic patients, which may explain why an association between TLG and CTCs was observed. In a comparable group of NSCLC patients undergoing surgery, presence of CTCs one month after resection correlated with SUV max , but TLG was not analyzed in this study (17). In a heterogeneous group of NSCLC patients of all stages, CTCs detected by a non-EpCAM-based method neither correlated with SUV max , nor TLG (18). Interestingly, presence of CTC clusters (≥2 CTCs in aggregate) was associated with TLG, but it needs to be taken into consideration that clusters are more prevalent in metastatic NSCLC (21). EpCAM-independent detection technologies yield higher CTC counts due to down-regulation of EpCAM during epithelial-mesenchymal transition of cancer cells (18). However, we chose to identify CTCs as CK pos /EpCAM pos -in alignment with the FDAapproved definition (22).
This pilot study links TLG to CTCs in non-metastatic NSCLC patients. Although we exclusively studied patients with local tumors or loco-regional nodal disease (without distant metastases), the extent of tumor burden varied amongst patients which may affect glucose metabolism and CTC shedding. Interpretation of our results are limited by small sample size and should be confirmed in larger cohorts.

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F-FDG PET/CT TLG, in contrast to SUVs, is associated with presence of CTCs in stage I-IIIA NSCLC patients. TLG might be an appropriate marker than SUVs for micrometastatic spread of NSCLC measured by CTCs in the blood.

Acknowledgments
We are very grateful to all subjects and their families for their voluntary participation, and to Blanche L. Lasta, RN, and Ken Baker, RN, for assistance in trial conductance. Funding: None.

Footnote
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi. org/10.21037/tlcr.2020.04.10). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Institutional Review Board of University of Missouri (IRB2010166/ IRB2004401-VA) and informed consent was taken from all individual participants.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the noncommercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.