Simple and robust diagnosis of early, small and AFP-negative primary hepatic carcinomas: an integrative approach of serum fluorescence and conventional blood tests

Abstract

The diagnosis of early, small and alpha-fetoprotein (AFP)-negative primary hepatic carcinomas (PHCs) remains a significant challenge. We developed a simple and robust approach to noninvasively detect these PHCs. A rapid, high-throughput and single-tube method was firstly developed to measure serum autofluorescence and cell-free DNA (cfDNA)-related fluorescence using a real-time PCR system, and both types of serum fluorescence were measured and routine laboratory data were collected in 1229 subjects, including 353 PHC patients, 331 liver cirrhosis (LC) patients, 213 chronic hepatitis (CH) patients and 332 normal controls (NC). The results showed that fluorescence indicators of PHC differed from those of NC, CH and LC to various extents, and all of them were not associated with age, gender, or AFP level. The logistic regression models established with the fluorescence indicators alone and combined with AFP, hepatic function tests and blood cell analyses were valuable for distinguishing early, small, AFP-negative and all PHC from LC, CH, NC and all non-PHC, with areas under the receiver operating characteristic curves 0.857-0.993 and diagnostic accuracies 80.2-97.7%. Conclusively, serum autofluorescence and cfDNA-related fluorescence are able to be rapidly and simultaneously measured by our simple method and valuable for diagnosing early, small and AFP-negative PHCs, especially integrating with AFP and conventional blood tests.

Keywords: AFP-negative hepatoma; diagnostic model; early diagnosis; primary hepatic carcinoma; serum fluorescence.

Figure 1. Optimum conditions for the fluorescence intensity measurements of pooled serum samples

(A) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to pooled serum volumes at 37°C (T37). (B) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to detection temperatures for 3 μL (S3) and 15 μL (S15) of pooled serum. (C, D) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to EvaGreen volumes for 3 μL (S3) and 15 μL (S15) of pooled serum at 8°C (T8) and 37°C (T37). (E, F) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to the cycle numbers (incubation time) for 3 μL of pooled serum at 8°C (S3T8) and 15 μL of pooled serum at 37°C (S15T37) in the absence of EvaGreen and for 3 μL of pooled serum at 8°C (S3T8E) and 15 μL of pooled serum at 37°C (S15T37E) in the presence of EvaGreen. PHC: primary hepatic carcinoma; LC: liver cirrhosis; CH: chronic hepatitis; NC: normal control.

Figure 2. The serum fluorescence intensities of the PHC, LC, CH and NC groups

(A–E) The comparisons of different sub-groups of fluorescence indicators between PHC and LC, CH or NC. *P < 0.05, **P < 0.01, compared with the PHC group by multiple comparisons (Dunnett's T3 test) after ANOVA. PHC: primary hepatic carcinoma; LC: liver cirrhosis; CH: chronic hepatitis; NC: normal control. The names of the fluorescence indicators are combinations of several abbreviations representing the fluorescence intensity (F) of 3 μL (S3) or 15 μL (S15) of serum at a detection temperature of 8°C (T8) or 37°C (T37) in the presence of EvaGreen (E). Moreover, the names indicate differences for two indicators between 3 μL and 15 μL (SD) of serum, for two temperatures of 8°C and 37°C (TD) and for the presence or absence of EvaGreen (ED).

Figure 3. The comparisons of 8 original fluorescence indicators between PHC sub-groups based on serum AFP level

(A), BCLC stage (B), tumor size (C), and tumor histology (D). *P < 0.05, **P < 0.01, compared between two subgroups. AFP: alpha-fetoprotein; BCLC: Barcelona Clinic Liver Cancer stage; HCC: hepatocellular carcinoma; ICC: intrahepatic cholangiocarcinoma; PHC: primary hepatic carcinoma. The name of each fluorescence indicator is a combination of several abbreviations representing the fluorescence intensity (F) of 3 μL (S3) or 15 μL (S15) of serum at a detection temperature of 8°C (T8) or 37°C (T37) in the presence (E) or absence of EvaGreen.

Figure 4. Diagnostic value of the models for diagnosing PHC vs. NC

(A), LC (B), CH (C) and NPHC (D). AUROC: area under the receiver operating characteristic curve; CI: confidence interval; PHC: primary hepatic carcinoma; NC: normal control; LC: liver cirrhosis; CH: chronic hepatitis; NPHC: non-primary hepatic carcinoma (NC+LC+CH). Each model name is a combination of abbreviations representing fluorescence intensity (F), alpha-fetoprotein (A), hepatic function tests (H) and/or blood cell analyses (B) with the model (−M), which indicates the covariates used during modeling; for example, FAHB-M was established with indicators of fluorescence intensity, alpha-fetoprotein, hepatic function tests and blood cell analyses. SEN: sensitivity; SPE: specificity; ACC: accuracy; PPV: positive predictive value; NPV: negative predictive value; PLR: positive likelihood ratio; NLR: negative likelihood ratio.

Figure 5. Diagnostic value of the models for diagnosing AFP-negative PHC vs. NC

(A), LC (B), CH (C) and NPHC (D). AUROC: area under the receiver operating characteristic (ROC) curve; CI: confidence interval; ANPHC: AFP-negative primary hepatic carcinoma; NC: normal control; LC: liver cirrhosis; CH: chronic hepatitis; NPHC: non-primary hepatic carcinoma (NC+LC+CH). Each model name is a combination of abbreviations indicating fluorescence intensity (F), alpha-fetoprotein (A), hepatic function testresults (H) and blood cell analyses (B) with the model (−M), which indicate the covariates used during modeling; for example, FAHB-M was established with indicators of fluorescence intensity, alpha-fetoprotein, hepatic function test results and blood cell analyses. SEN: sensitivity; SPE: specificity; ACC: accuracy; PPV: positive predictive value; NPV: negative predictive value; PLR: positive likelihood ratio; NLR: negative likelihood ratio.

Figure 6. Diagnostic value of the models for diagnosing PHC at BCLC stage A vs. NC

(A), LC (B), CH (C) and NPHC (D). AUROC: area under the receiver operating characteristic (ROC) curve; CI: confidence interval; BCLC-A: Barcelona Clinic Liver Cancer stage A; PHC: primary hepatic carcinoma; NC: normal control; LC: liver cirrhosis; CH: chronic hepatitis; NPHC: non-primary hepatic carcinoma (NC+LC+CH). Each model name is a combination of abbreviations representing fluorescence intensity(F), alpha-fetoprotein (A), hepatic function test results (H) and/or blood cell analyses (B) with the model (−M), which indicate the covariates used during modeling; for example, FAHB-M was established with indicators of fluorescence intensity, alpha-fetoprotein, hepatic function test results and blood cell analyses. SEN: sensitivity; SPE: specificity; ACC: accuracy; PPV: positive predictive value; NPV: negative predictive value; PLR: positive likelihood ratio; NLR: negative likelihood ratio.

Figure 7. Diagnostic value of the models for discriminating small PHC from NC

(A), LC (B), CH (C) and NPHC (D). AUROC: area under the receiver operating characteristic (ROC) curve; CI: confidence interval; SPHC: small primary hepatic carcinoma; NC: normal control; LC: liver cirrhosis; CH: chronic hepatitis; NPHC: non-primary hepatic carcinoma (NC+LC+CH). Each model name is a combination of abbreviations representing fluorescence intensity (F), alpha-fetoprotein (A), hepatic function tests (H) and/or blood cell analyses (B) with the model (−M), which indicate the covariates used during modeling; for example, FAHB-M was established with indicators of fluorescence intensity, alpha-fetoprotein, hepatic function tests and blood cell analyses. SEN: sensitivity; SPE: specificity; ACC: accuracy; PPV: positive predictive value; NPV: negative predictive value; PLR: positive likelihood ratio; NLR: negative likelihood ratio.

Figure 8. Diagram of the measurement of serum autofluorescence and cell-free DNA-related fluorescence

FS3T8, FS15T8, FS3T37, FS15T37, FS3T8E, FS15T8E, FS3T37E and FS15T37E: the names of 8 original fluorescence indicators. Each indicator name is an abbreviation indicating the fluorescence intensity (F) of serum (S) 3 μL (3) or 15 μL (15) at a given temperature (T) 8°C (8) or 37°C (37) in the presence (E) or absence of EvaGreen.