Assessment of the Mutational Status of NSCLC Using Hypermetabolic Circulating Tumor Cells

Abstract

Molecular characterization is currently a key step in NSCLC therapy selection. Circulating tumor cells (CTC) are excellent candidates for downstream analysis, but technology is still lagging behind. In this work, we show that the mutational status of NSCLC can be assessed on hypermetabolic CTC, detected by their increased glucose uptake. We validated the method in 30 Stage IV NSCLC patients: peripheral blood samples were incubated with a fluorescent glucose analog (2-NBDG) and analyzed by flow cytometry. Cells with the highest glucose uptake were sorted out. EGFR and KRAS mutations were detected by ddPCR. In sorted cells, mutated DNA was found in 85% of patients, finding an exact match with primary tumor in 70% of cases. Interestingly, in two patients multiple KRAS mutations were detected. Two patients displayed different mutations with respect to the primary tumor, and in two out of the four patients with a wild type primary tumor, new mutations were highlighted: EGFR p.746_750del and KRAS p.G12V. Hypermetabolic CTC can be enriched without the need of dedicated equipment and their mutational status can successfully be assessed by ddPCR. Finally, the finding of new mutations supports the possibility of probing tumor heterogeneity.

Circulating Tumor Cells in Diagnosis and Treatment of Lung Cancer

“…2-NBDG) for a short time and then be evaluated using fluorescence microscopy. Turetta et al have shown that glucose uptake values of CTCs are a median of 10 times higher than those of white blood cells (44).…”

Dysmetabolic Circulating Tumor Cells Are Prognostic in Metastatic Breast Cancer

“…A plethora of new technologies emerged to overcome the limitations of EpCAM-based approaches, focusing on the challenge of detecting non-epithelial CTCs, mainly exploiting mesenchymal markers or physical features of cancer cells and other cancer-specific traits. Notably, few groups have specifically addressed altered metabolism of cancer cells [22][23][24][25][26][27]-recognized as a "hallmark of cancer" [28]-as a promising approach, also in view of the introduction of specific metabolic agent combined with chemo-or immunotherapies in several clinical trials [29][30][31]. One of the most described metabolic alterations of cancer cells consists of a markedly increased uptake of glucose in comparison with normal cells.…”

“…First described by Otto Warburg in the 1920s, this cancer feature has been successfully exploited in the clinic by introducing a positron emission tomography (PET)-based imaging of the uptake of a radioactive glucose analog, which is commonly employed to stage cancer and assess response to therapy [32]. Several groups reported the exploitation of metabolic alterations to identify CTCs in the peripheral blood of metastatic cancer patients, independently from EpCAM expression and immunostaining techniques in general [22][23][24][25][26][27]. An mRNAseq study on CTCs from prostate cancer patients described a category of CTCs with an upregulation of metabolic transcripts as a potential biomarker for cancer metastasis [24].…”

Dysmetabolic circulating tumor cells are prognostic in metastatic breast cancer

“…A plethora of new technologies emerged to overcome the limitations of EpCAM-based approaches, focusing on the challenge of detecting non-epithelial CTCs, mainly exploiting mesenchymal markers or physical features of cancer cells and other cancer-specific traits. Notably, few groups have specifically addressed altered metabolism of cancer cells (22)(23)(24)(25)(26)(27) recognized as a "hallmark of cancer"(28) -as a promising approach, also in view of the introduction of specific metabolic agent combined with chemo-or immunotherapies in several clinical trials (29)(30)(31).…”

“…First described by Otto Warburg in the 1920s, this cancer feature has been successfully exploited in the clinic by introducing a positron emission tomography (PET)-based imaging of the uptake of a radioactive glucose analog, which is commonly employed to stage cancer and assess response to therapy (32). Several groups reported the exploitation of metabolic alterations to identify CTCs in the peripheral blood of metastatic cancer patients, independently from EpCAM expression and immunostaining techniques in general (22)(23)(24)(25)(26)(27). An mRNAseq study on CTCs from prostate cancer patients described a category of CTCs with an upregulation of metabolic transcripts as a potential biomarkers for cancer metastasis (24).…”