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. 2024 Dec 18;16(24):4220.
doi: 10.3390/cancers16244220.

Evaluation of Thermal Liquid Biopsy Analysis of Saliva and Blood Plasma Specimens as a Novel Diagnostic Modality in Head and Neck Cancer

Gabriela Schneider  1 , Alagammai Kaliappan  1 , Nathan Joos  2 , Laura M Dooley  2 , Brian S Shumway  3 , Jonathan B Chaires  1   4 , Wolfgang Zacharias  1   4 , Jeffrey M Bumpous  2 , Nichola C Garbett  1   4
Affiliations

Evaluation of Thermal Liquid Biopsy Analysis of Saliva and Blood Plasma Specimens as a Novel Diagnostic Modality in Head and Neck Cancer

Gabriela Schneider et al. Cancers (Basel). .

Abstract

Background: Over the past decade, saliva-based liquid biopsies have emerged as promising tools for the early diagnosis, prognosis, and monitoring of cancer, particularly in high-risk populations. However, challenges persist because of low concentrations and variable modifications of biomarkers linked to tumor development when compared to normal salivary components.

Methods: This study explores the application of differential scanning calorimetry (DSC)-based thermal liquid biopsy (TLB) for analyzing saliva and blood plasma samples from head and neck cancer (HNC) patients.

Results: Our research identified an effective saliva processing method via high-speed centrifugation and ultrafiltration, resulting in reliable TLB data. Notably, we recorded unique TLB profiles for saliva from 48 HNC patients and 21 controls, revealing distinct differences in thermal transition features that corresponded to salivary protein denaturation. These results indicated the potential of saliva TLB profiles in differentiating healthy individuals from HNC patients and identifying tumor characteristics. In contrast, TLB profiles for blood plasma samples exhibited smaller differences between HNC patients and had less utility for differentiation within HNC.

Conclusions: Our findings support the feasibility of saliva-based TLB for HNC diagnostics, with further refinement in sample collection and the incorporation of additional patient variables anticipated to enhance accuracy, ultimately advancing non-invasive diagnostic strategies for HNC detection and monitoring.

Keywords: TLB profile; blood plasma; diagnosis; diagnostic modality; differential scanning calorimetry (DSC); head and neck cancer (HNC); saliva; thermal liquid biopsy (TLB).

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Conflict of interest statement

N.C.G. is a co-inventor on patent/patent applications assigned to and owned by the University of Louisville. U.S. Patent Application No. 15/764,458 describes approaches for the analysis of DSC plasma thermogram data and their use for diagnostic classification; U.S. Patent No. 11,835,529 B1 describes the use of DSC for diagnosis and treatment of acute myocardial infarction; and U.S. Patent Application No. 17/771,487 describes a microfabricated DSC system. N.C.G. and G.S. are co-inventors on a patent application assigned to and owned by the University of Louisville describing the passive isolation of blood plasma from a whole blood sample (U.S. Patent Application No. 17/080,805). During study completion and analysis, N.C.G. and G.S. were founders and had an equity interest in a start-up company, DSC Technologies LLC, which was involved in the development of DSC technologies; N.C.G. was a consultant for the calorimetry instrument supplier TA Instruments, Inc., involved in education for microcalorimetry applications and the characterization of microcalorimetry instrument performance. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The declared conflicts of interest do not alter the authors’ adherence to all of Cancers’ policies on sharing data and materials.

Figures

Figure 1
Figure 1
Schematic representation of the possible use of DSC-based TLB of saliva and/or plasma for screening, diagnosis, prognosis, or monitoring of HNC patients. The process begins with the collection of saliva and/or blood from patients, followed by saliva processing and/or blood plasma isolation. TLB involves DSC analysis to capture the comprehensive protein denaturation behavior of the patient saliva/plasma samples. The resulting TLB profiles are used in the detection of HNC and for further characterization of HNC for personalized monitoring of HNC patients.
Figure 2
Figure 2
The impact of the saliva preparation method on TLB profiles. (A) TLB profiles of saliva samples processed using different methods incorporating centrifugation and/or filtration. (B) TLB profiles of saliva samples prepared using methods incorporating acid precipitation of samples. TLB profiles obtained using Method 2, identified as the most optimal processing method, is used for comparison. (C) TLB profiles of individual proteins abundant in saliva.
Figure 3
Figure 3
Comparison of TLB profiles of saliva samples obtained from healthy volunteers (Controls) and HNC patients. (A) Plot of the mean TLB profile value at each temperature for Controls (n = 21) and HNC patients (n = 48). (B) Boxplots of metrics and PCs calculated from TLB profiles for Controls and HNC patients. Unadjusted p-values < 0.05 are shown on the graphs.
Figure 4
Figure 4
Comparison of TLB profiles of saliva samples obtained from healthy volunteers (Controls) and HNC patients separated into groups based on overall cancer stage. (A) Plot of the mean TLB profile value at each temperature for Controls (n = 21) and patients with different overall stages of HNC (Stage I, n = 4; II, n = 10; III, n = 15; IV, n = 19). (B) Boxplots of metrics and PCs calculated from TLB profiles for Controls and patients with different overall stages of HNC. Unadjusted p-values < 0.05 are shown on the graphs.
Figure 5
Figure 5
Comparison of TLB profiles of saliva samples obtained from healthy volunteers (Controls) and HNC patients separated into groups based on the cancer location. (A) Plot of the mean TLB profile value at each temperature for Controls (n = 21) and patients with different locations of HNC (FOM, n = 5; Tongue, n = 16; Tonsil, n = 18; Other, n = 9). (B) Boxplots of metrics and PCs calculated from TLB profiles for Controls and patients with different locations of HNC. Unadjusted p-values < 0.05 are shown on the graphs.
Figure 6
Figure 6
Comparison of TLB profiles of plasma samples obtained from HNC patients separated into groups based on the overall cancer stage. (A) Plot of the mean TLB profile value at each temperature for patients with different overall stages of HNC (Stage I, n = 4; II, n = 10; III, n = 15; IV, n = 19). (B) Boxplots of metrics and PCs calculated from TLB profiles for patients with different overall stages of HNC. Unadjusted p-values < 0.05 are shown on the graphs.
Figure 7
Figure 7
Comparison of TLB profiles of plasma samples obtained from HNC patients separated into groups based on the cancer location. (A) Plot of the mean TLB profile value at each temperature for patients with different locations of HNC (FOM, n = 5; Tongue, n = 16; Tonsil, n = 18; Other, n = 9). (B) Boxplots of metrics and PCs calculated from TLB profiles for patients with different locations of HNC. No significant differences in metrics or PCs were observed.
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