Blood samples of consecutive patients with locally advanced, recurrent, or metastatic malignant diseases were taken in our clinical chemistry laboratory (Szent Lázár County Hospital, Salgótarján, Hungary) as part of the routine investigation before the initiation of the therapy of the given disease. Obvious symptoms and signs of common infectious diseases were assessed (purulent cough, pulmonary crackles, or symptomatic bacteriuria). Exclusion criteria included suspected infection, hematological malignancy, the lack of at least one biomarker data point, rapid progression (i.e., from laboratory testing, ECOG performance status progressed to 3 before the initiation of anticancer treatment), or death caused by something other than disease progression. Patients with all the following biomarkers available were included in the study: CRP, D-dimer, LDH, albumin, and complete blood count (CBC). Data of 13 excluded patients are given in Supplementary File S1.

CRP, LDH, and albumin were measured with commercially available Roche tests on Cobas c501 or Cobas 6000 analyzers (Tokyo, Japan). D-dimer levels were measured by a chemiluminescent immunoassay (PATHFAST, Tokyo, Japan). CBC was determined with Cell-dyn 3700 (Abbott Park, IL, United States and Beckman Unicel DxH600, Miami, FL, United States). The LMR, NLR, and PLR were calculated as the ratio of the lymphocyte count and the monocyte count, the ratio of the neutrophil count and the lymphocyte count, and the ratio of the platelet count and the lymphocyte count, respectively.

For the purpose of statistical analysis, we used for CRP <5 mg/L (lower level of detection), the value of 4.9 mg/L, and for D-dimer >5 mcg/mL (higher level of detection), the value of 5.1 mcg/mL. All other biomarker values were handled with the measured numeric values. Cut-off determination was performed with the validated “Cutoff Finder” online tool (46). After uploading the tab separated value file (Supplementary File S2), for each biomarker the “Survival Time” was OS or censored OS, the “Survival Event” was the variate of 1 for OS or 0 for censored OS, and the “Method for cut-off determination” was “Survival: significance (log-rank test). Statistical analysis was performed by R Studio Software (47). Semicolon separated value file (Supplementary File-2b.csv) was used.” For each value a comparison was made between the median OS values below and over the cut-off value by the log-rank test. The value with the largest gap and Chi-squared statistics was selected. Comparison of the prognostic groups with Cox proportional hazard regression was performed. Log-rank test was used to detect the differences between survival curves within the prognostic groups in the Kaplan-Meier analysis as well as to assess the significance of the Cox model. Effect size estimation was performed for the Mann-Whitney probe by calculating the so called Eta-squared value. Between 0.06 and 0.14, the effect can be considered medium-sized, while over 0.14 it can be considered large. Power analysis was performed with the “powerCT” function in the “powerSurvEpi” package of the R Studio software. All figures were drawn as vector graphics in Scalable Vector Graphic format in the “ggsurvplot” and “ggforest” functions in the “survminer” package of the R Studio software (47) and edited by Inkscape software (https://inkscape.org). The R-script is available in Supplementary File S3.

OS time was defined as the length of survival from the date of laboratory testing. Survival data measured in months were computed according to Surveillance, Epidemiology, and End Results (SEER) recommendations (https://seer.cancer.gov/survivaltime/SurvivalTimeCalculation.pdf): days between the dates were divided by one twelfth of 365.24. For the median follow-up time calculation, we used a reverse Kaplan-Meier estimator (48).

Between July 2016 and August 2019, blood samples of 88 consecutive patients with locally advanced, recurrent, or metastatic malignant disease were analyzed. No common infectious diseases were diagnosed. Data of 13 patients were excluded from the final analysis because of hematological malignancy 1), the lack of any biomarker data (2), death caused by rapid progression before the initiation of anticancer therapy (4), or by other cause of death than disease progression (6). Thus the final retrospective analysis included the data of 75 patients. The shortest censored survival time was 24 months, i.e., the time elapsed since July 2019. As of July 2021, six (8%) patients were still alive. Data of patient characteristics are described in Table 1. Additional data are given in Supplementary Tables S1–S11 in Supplementary File S1.

The Kaplan-Meier plot was used to determine the median OS and the median follow-up times. With a median follow-up of 46.98 months [95% confidence interval (CI): 37.16–49.28] the median OS was 12.12 months (95% CI: 7.85–18.33) (Figure 1). Mean values of CRP, D-dimer, LDH, albumin, LMR, NLR, and PLR were 28.83 mg/L, 1.70 mcg/mL, 482.12 U/L, 41.62 g/L, 3.41, 4.29, and 168.83, respectively.

With the combination of three biomarkers, prognostic groups were created independently from stage, histology, and time to progression on first line therapy (Supplementary File S1). Four prognostic groups were formed based on the cut-off values of each biomarker. Group 1: No biomarker with out-of-range value (ORV), defined by the cut-off value; group 2: One ORV biomarker; group 3: Two ORV biomarkers; and group 4: Three ORV biomarkers (Table 3). Significant differences were detected between these groups (Table 4, Figure 3). The likelihood ratio test of the Cox model regression parameters for the four groups was 29.5 (p < 0.001).

We compared the median OS of groups with one ORV biomarker with that of groups with two and three ORV biomarkers using the Mann-Whitney test and Z statistic (Table 5). The comparison of the group of ORV albumin with the group of ORV CRP and albumin values indicated a significant difference (p = 0.04; Eta-squared = 0.067; medium power size). A similar significance was detected comparing the group ORV albumin with the group of ORV CRP and PLR (p = 0.026; Eta-squared = 0.087; medium power size). The cases in the groups of ORV CRP and PLR also had ORV albumin.

No significant differences were found between the groups with two ORV biomarkers with three ORV biomarkers.