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Article

Immune Marker and C-Reactive Protein Dynamics and Their Prognostic Implications in Modulated Electro-Hyperthermia Treatment in Advanced Pancreatic Cancer: A Retrospective Analysis

by
Nikolett Kitti Dobos
1,
Tamas Garay
1,2,
Magdolna Herold
2,3,
Alexandra Simon
1,
Viktor Madar-Dank
4,
Gyula Balka
5,
Jozsef Gajdacsi
6,
Magdolna Dank
2,
Attila Marcell Szasz
2,* and
Zoltan Herold
2,*
1
Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, H-1083 Budapest, Hungary
2
Division of Oncology, Department of Internal Medicine and Oncology, Semmelweis University, H-1083 Budapest, Hungary
3
Department of Internal Medicine and Hematology, Semmelweis University, H-1088 Budapest, Hungary
4
Department of Finance, Rutgers University, Newark, NJ 07102, USA
5
Department of Pathology, University of Veterinary Medicine Budapest, H-1078 Budapest, Hungary
6
Clinical Center, Semmelweis University, H-1083 Budapest, Hungary
*
Authors to whom correspondence should be addressed.
Immuno 2024, 4(4), 385-399; https://doi.org/10.3390/immuno4040025
Submission received: 30 August 2024 / Revised: 9 October 2024 / Accepted: 16 October 2024 / Published: 18 October 2024
(This article belongs to the Section Cancer Immunology and Immunotherapy)
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Abstract

Background: Previous research has suggested that modulated electro-hyperthermia (mEHT) can be used to induce anti-tumor immune effects and to extend patient survival. The use of mEHT in advanced pancreatic cancer is beneficial; however, its immune-mediating effects were never investigated. Methods: A retrospective observational study was conducted. Leukocyte counts, C-reactive protein (CRP), neutrophil-to-lymphocyte ratio (NLR), and granulocyte-to-lymphocyte ratio (GLR) were measured at baseline, midpoint, and after mEHT treatment. Results: A total of 73 mEHT treated pancreatic cancer patients were included. The time elapsed between tumor diagnosis and the first mEHT treatment was 4.40 ± 5.70 months. While no change could be observed between the baseline and the first follow-up visits, the total white blood cell (WBC), neutrophil, and granulocyte count, CRP, NLR, and GLR were significantly higher at the second follow-up compared to both previous visits. Higher levels of the latter parameters following the last mEHT treatment were signaling significantly poor prognostic signs, and so were their longitudinal changes. Conclusions: After the initiation of mEHT, immune markers stabilize with the treatment, but this positive effect is eroded over time by progressive disease. Monitoring the changes in these markers and the occurrence of their increase is a prognostic marker of shorter survival.

1. Introduction

According to the data of GLOBOCAN, more than half a million patients were diagnosed with pancreatic cancer in 2022 globally. The incidence of the disease is nearly identical with its mortality rate [1]. It is predicted that pancreatic cancer is going to be the second leading cause of cancer-related deaths by 2030 in the United States [2]. In Hungary, 2700–2800 new cases are registered annually [3]. Pancreatic ductal adenocarcinoma (PDAC) comprises over 90% of pancreatic tumors, with a 5-year survival rate of less than 10% [4]. Approximately 80% of patients are diagnosed with advanced stages, implying the presence of local and possibly distant metastases [5]. In advanced cases, the patients are usually not eligible for tumor resection [6]. The conventional therapeutic option is systemic chemotherapy with or without radiotherapy. The standard of care in PDAC, if the patient’s performance status allows it, can be the FOLFIRINOX protocol (a combination of fluorouracil, leucovorin, irinotecan, and oxaliplatin), or gemcitabine combined with platinum-based agents or nab-paclitaxel [4,5]. However, PDACs are resistant against most of the therapeutical arsenal [4]. The median overall survival of PDAC patients on gemcitabine monotherapy was previously 4–6 months [7,8,9]; however, with the introduction of FOLFIRINOX, gemcitabine with nab-paclitaxel, and concomitant radiotherapy, it moderately raised to 8–12 months [10,11,12]. The incidence of PDAC and PDAC-related death are expected to rise [9].
Recent research suggests that PDAC patients with microsatellite instability could be treated with immune checkpoint inhibitors (such as nivolumab, ipilimumab, tocilizumab, and pembrolizumab) as first-line immunotherapy. It is feasible, however, for the minority (~2%) of PDAC patients only. PDAC is characterized by an immunosuppressive microenvironment. The evasion of the immune system is related to the poor prognosis of the disease. Other new immunotherapies have yet to be investigated due to them being in the experimental phase [13]. As the disease is highly resistant against a wide range of therapies, new mechanisms of actions and more effective therapy regimens must be developed, while improving the quality of life of the patients. To overcome this, in previous decades, several multimodal therapies emerged to complement tumor resection and chemoradiotherapy including thermal ablation techniques and hyperthermia [14].
In oncology, concomitant hyperthermia refers to the heating of the tumor tissue to 39–42 °C in a local, regional, or systemic manner [15]. Hyperthermia results in biophysical changes that directly alter cellular metabolism and initiate an anti-tumor response by immunomodulation [16]. Modulated electro-hyperthermia (mEHT), one of the latest advancements in hyperthermia, is a novel precision method that employs capacitive coupling and impedance matching [14,17]. Although mEHT is a form of hyperthermia in its name, its mechanism of action resembles electromagnetic therapies. The biological principle of mEHT is based on the differential membrane permeability and conductivity of cancer cells relative to healthy cells. In response to the treatment, the membrane rafts of tumor cells undergo specific energy absorption, which results in the production of danger-associated molecular patterns (DAMPs). Ultimately, the process leads to programmed or immunogenic cell death [16,17]. The tumor-cell-selective local approach results in several other effects that are favorable in the context of tumor therapy: the triggering of the heat shock response, immunological stimulation, altered perfusion, and enhanced drug delivery are only a few instances of the benefits. In refraction, mEHT enables the re-sensitization of tumor cells, thereby enhancing the effectiveness of parallel treatments, as well as the overall survival and quality of life [18].
Over the past decade, only a limited number of studies have explored the effects of mEHT/hyperthermia on the immune system in cancer patients. One study involving 97 patients with refractory advanced non-small-cell lung cancer have found that treatment with mEHT and vitamin C has reduced the serum level of C-reactive protein (CRP), while significantly improving the overall survival and the quality of life of the patients [19]. Furthermore, two studies have reported that local hyperthermia positively influences the immune response in patients with hepatocellular carcinoma [20,21]. Moreover, some evidence suggests that this treatment may positively affect the immune system in patients with glioblastoma and cervical cancer [22,23]. However, no studies have specifically investigated the impact of mEHT on routinely measured immunological markers. Therefore, the aim of this retrospective observational cohort study was to examine the longitudinal effects of concomitant mEHT on the complete leukocyte count and CRP levels in patients with advanced- and late-stage PDAC.

2. Materials and Methods

This study was approved by the Regional and Institutional Committee of Science and Research Ethics, Semmelweis University (SE TUKEB 8/2017, SE TUKEB 8-1/2017; approval date of the latest version: 9 January 2023). This study was conducted in accordance with the WMA Declaration of Helsinki. Patient data were handled in accordance with the General Data Protection Regulation issued by the European Union. The patients treated with mEHT provided their written consent to participate in this study.

2.1. Study Design and Patient Selection

A retrospective observational cohort study was performed. A total of 73 advanced- and late-stage PDAC patients treated at the Division of Oncology, Department of Internal Medicine and Oncology, Semmelweis University, Budapest, Hungary, between September 2016 and March 2021 were included. All patients received standard-of-care chemotherapy and concomitant mEHT treatment. Patients under the age of 18, being treated for an active-phase autoimmune disease or an active infection such as hepatitis B, diagnosed with non-adenocarcinoma-type pancreatic cancer, mental illnesses, inflammatory bowel diseases (e.g., ulcerative colitis or Chron’s disease), untreated thyroid diseases, and a ≥2 Eastern Cooperative Oncology Group (ECOG) performance score were excluded from the study. An additional requirement for the study was that all patients had to have complete blood count and CRP results at least within 30 days before (baseline measurement) the mEHT treatment. In addition, there were two follow-up measurements in the middle of mEHT treatment and afterwards, in which laboratory findings were required for at least one of them.

2.2. Modulated Electro-Hyperthermia Treatment

Concomitant mEHT treatments were carried out using the Oncotherm EHY-2000+ and the EHY-2030 (Oncotherm Kft., Budaörs, Hungary) devices. Both devices use the same technological principle and are comparable. The main difference between the two lies in user friendliness and convenience changes. Patients received treatment twice or three times a week, in a lying position. The duration of the sessions was 60 min. During the first week, treatments were conducted at a lower power setting, gradually increasing the device’s power from 60 to 100 W over a 30 min period, and then maintaining that level until the end of the session. From the second week, the power was increased from 100 to 150 W over 30 min and sustained at that level for the remaining 30 min. If any adverse reactions occurred during the power increase, the process was halted, and the treatment was continued at the highest tolerable power. If any adverse effects (e.g., skin redness) were observed, the session was terminated. Except for skin redness in a few cases, no adverse reactions were noted during this study.

2.3. Clinical Characteristics

Disease history data and laboratory results of fasting blood samples were collected and recorded for every visit. Treatment and measurement details were collected from the hospital information system (e-MedSolution, Egészséginformatikai Szolgáltató és Fejlesztési Központ, Budapest, Hungary) of Semmelweis University. Complete blood count and C-reactive protein level were determined at the Central Laboratory of Semmelweis University, Budapest, Hungary. In addition, the neutrophil-to-lymphocyte ratio (NLR) was calculated as an immunological tumor marker [24]. In the case of missing neutrophil granulocyte count data, the total granulocyte-to-lymphocyte ratio (GLR) was used [25]. All patients were treated with standard-of-care chemotherapy regimens, based on national and international guidelines [26,27]. Most of the patients were treated with FOLFIRINOX, gemcitabine ± nab-paclitaxel, or mono-capecitabine. The location of the tumors was classified as head, body, and tail [8]. The dates of the first and last mEHT treatments were recorded. The overall survival (OS) of the patients was defined as the duration between the first mEHT treatment and the death of the patient. Additionally, survival between the last mEHT treatment and the death of the patient was also calculated. The follow-up was terminated on 30 June 2024. Patients alive at this time point were right-censored.

2.4. Statistical Analysis

Statistical analysis was carried out using the R for Windows version 4.4.0 environment (R Foundation for Statistical Computing, 2024, Vienna, Austria). Comparison of sub-cohorts was performed using the Wilcoxon rank sum test and Fisher’s exact test. Laboratory parameters measured at the three timepoints were compared using the Wilcoxon signed-rank tests and linear mixed-effects models (R package nlme, version 3.1-165). To examine overall survival and calculate hazard ratios (HR) with 95% confidence intervals (95% CI), basic and extended Cox proportional hazards regression models with time-dependent coefficients were used (R package survival, version 3.7-0). If the proportional hazard assumption was violated, the survival models were adjusted with step functions, following the method outlined by Therneau et al. [28]. Receiver operating curve (ROC) analysis with Youden’s J statistic was used to obtain optimal cut-off values of the investigated parameters [29]. Two-sided p-values < 0.05 were considered as significant, and all p-values were corrected for the multiple comparisons problem using the Holm method [30]. Continuous data, frequencies, and survival data were expressed as mean ± SD, number of observations (percentages), and HR with its 95% CI, respectively. Naïve Kaplan–Meier curves were generated using the “survminer” R package (version 0.4.9).

3. Results

The retrospective data of the 73 advanced- and late-stage PDAC patients were collected to assess the effects of mEHT treatment. The baseline characteristics of the patients are summarized in Table 1. The relationship between the number of mEHT treatment cycles and baseline clinicopathological data was assessed: no connection and/or correlation could be proven with any of them.

3.1. Comparison of the Leukocyte Counts, CRP Levels, and NLR and GLR Values Measured at the Baseline and Follow-Up Visits

Leukocyte counts and their ratios, and CRP, were collected for three timepoints during the study: (1.) at baseline, 6 ± 10 days prior to mEHT treatment, (2.) at the midpoint of the mEHT treatments (on average 67 ± 44 days after the first mEHT treatment), and (3.) 16 ± 21 days after the last mEHT treatment. To analyze the longitudinal data, we chose two approaches using Wilcoxon signed-rank tests and linear mixed-effects models. First, the differences of the investigated parameter levels were evaluated at the three time points using Wilcoxon signed-rank tests (Table S1, Figure S1). It must be noted that, with this method, only the complete cases (where all three measurements were available) could be investigated. A significant decrease in the eosinophil granulocyte count was observed (p = 0.0412) upon the initialization of the mEHT treatment. Regarding the difference between the baseline measurement and the second follow-up, a significant increase was found in the total white blood cell count (WBC; p = 0.0426), the monocyte count (p = 0.0029), and the NLR value (p = 0.0152), while the eosinophil granulocyte count was found to be significantly lower (p = 0.0062). Between the two follow-ups, significant increases were observed in WBC (p = 0.0426), GLR (p = 0.0241), and NLR (p = 0.0292). Although the neutrophil granulocyte counts exhibited a similar trend to the WBC, the results failed to reach significance.
To further investigate the effect of mEHT treatment on the longitudinal data, linear mixed-effects models were applied. With this approach, not only the complete cases but all available measurements could be investigated. Similarly to that of the previous method, it was observed that the WBC, CRP, neutrophil count, granulocyte count, and NLR and GLR values were significantly higher in the last follow-up measurement, while at the first measurement no profound changes were observed (Figure 1 and Figure 2). No changes in the eosinophil, basophil, monocyte, and lymphocyte counts could be found (Table 2).

3.2. Survival Analyses

During the observational period of our study, except for a single female patient (~1.4%), basically the whole cohort deceased with a median survival of 13.24 months (if calculated from the time of the diagnosis). The median survival time from the first mEHT treatment was 8.15 months, and it was 4 months following the last mEHT treatment (Table 1). The OS of the whole cohort is presented in Figure S2. To determine the impact of the evaluated parameters on the survival of patients, the following analyses were completed. First, the baseline and first follow-up data were investigated, where the survival time was calculated from the first mEHT treatment. Second, the survival time was calculated from the date of the last mEHT treatment, and here, the second follow-up data were analyzed. Third, the longitudinal change in the parameters was investigated with extended Cox regression models. And, fourth, multivariate models were created to evaluate whether any of the parameters had superior prognostic value.

3.2.1. Survival After the Date of the First mEHT Treatment

An increased possibility of poor survival was observed with higher CRP levels measured at the first follow-up (HR: 1.0175; 95% CI: 1.0001–1.0352; p = 0.0482). Higher GLR at the middle of the mEHT treatments can anticipate a worse clinical outcome (HR: 1.0817; 95% CI: 0.9958–1.1749; p = 0.0629). Patients with tumors located in the head of the pancreas, as opposed to the tail, were associated with a lower risk of earlier death (HR: 0.4855; 95% CI: 0.2383–0.9891; p = 0.0466). Moreover, the occurrence of hepatic metastases was linked to an increased risk of mortality regardless of it being synchronous (HR: 2.3554; 95% CI: 1.3806–4.0184; p = 0.0017) or metachronous (HR: 1.8654; 95% CI: 0.9361–3.7172; p = 0.0764). Neither the remaining immune-related markers, age (p = 0.8995), sex (p = 0.1961), nor the presence of ascites (p = 0.9090) had any effect on the survival length of the patients. If the baseline hazards in the models were adjusted for tumor location and hepatic metastasis, the resulting clinically negative associations would remain essentially unchanged (CRP at first follow-up: p = 0.0910; GLR at first follow-up: p = 0.0663).

3.2.2. Survival After the Date of the Last mEHT Treatment

After the last mEHT treatment, a higher WBC count (HR: 1.0669; 95% CI: 1.0266–1.1087; p = 0.0010), neutrophil granulocyte count (HR: 1.0758; 95% CI: 1.0292–1.1245; p = 0.0012), total granulocyte count (HR: 1.0801; 95% CI: 1.0369–1.1251; p = 0.0002), and CRP level (HR: 1.0079; 95% CI: 1.0041–1.0118; p < 0.0001) were associated with a significantly higher risk of earlier death. The same was observed when the percentage distributions of white blood cell fractions were examined. Higher neutrophil (HR: 1.0456; 95% CI: 1.0195–1.0722; p = 0.0005) and total granulocyte percentages (HR: 1.0360; 95% CI: 1.0133–1.0593; p = 0.0018) are poor prognostic signs. In contrast, higher monocyte percentages indicated better survival (HR: 0.9302, 95% CI: 0.8711–0.9933, p = 0.0308). Additionally, the same observations were found as detailed previously, that longer survival could be expected if the tumor was in the head of the pancreas (HR: 0.4553; 95% CI: 0.2234–0.9276; p = 0.0302), and poorer survival was associated with the presence of synchronous hepatic metastases (HR: 2.3108; 95% CI: 1.3362–3.9960; p = 0.0027). Moreover, it was found that females tend to survive longer (HR: 0.6499; 95% CI: 0.4045–1.0441; p = 0.0748). The survival model results were not completely linear for the GLR and NLR data measured at the second follow-up. To obtain the correct results, the models had to be augmented with step functions, with which the observation period was split into two parts. For the first 3.5 months following the last mEHT treatment, both NLR (HR: 1,0740; 95% CI: 1.0390–1.1100; p < 0.0001) and GLR (HR: 1.0776; 95% CI: 1.0460–1.1100; p < 0.0001) were signs for poor prognosis. Similarly, both parameters indicated significantly worse prognosis for the period after 3.5 months (NLR: p = 0.0010; GLR: p = 0.0045), but their respective HRs were higher (NLR: 1.2584; GLR: 1.2127) compared to those of the first 3.5 months. Except for the tendentious differences between the two sexes, baseline hazard adjustment for tumor location and hepatic metastasis did not change any of the observations detailed in this paragraph. This latter observation (sex differences) was possibly related to the fact that hepatic metastases were more common in males (68.6% vs. 39.5%; p = 0.0384), and the tumor of the pancreatic head occurred somewhat more often in females (63.2% vs. 50.0%; p = 0.0945).

3.2.3. Do the Number of mEHT Treatment Cycles Affect Patient Survival?

The effect of mEHT treatment cycle numbers on survival after the first and after the last mEHT treatment were investigated. For the former, it was observed that, with administering a higher amount of mEHT treatments within the first 6 months after the first mEHT treatment, better survival of the patients could be achieved (HR: 0.9376; 95% CI: 0.9072–0.9690; p = 0.0001). However, no effect on patient survival could be justified for longer periods (6–15 months after the first mEHT treatment: p = 0.6381; more than 15 months after the first mEHT treatment: p = 0.2499). Similarly, the survival time length of patients was not affected by the number of mEHT treatment cycles if the survival was calculated from the date of the last mEHT treatment (p = 0.9975 and p = 0.8091 if adjusted for tumor location and presence of hepatic metastases).

3.2.4. Multivariate Survival Analyses

Multivariate models were created to assess the effect of more than one covariate on patient survival. The analyses were performed to assess the effects after both the first and last mEHT treatment dates. The following parameters were included in the models: last total WBC, GLR, or NLR value (corresponding to model 1, 2, and 3, respectively) with the last CRP measurement, mEHT treatment cycle count, tumor location, and the presence of hepatic metastases. It must be noted that, in the univariate models, no significance was obtained for ascites; however, if ascites was included in the multivariate models, it nearly outperformed virtually all of the other parameters. For this reason, ascites as a covariate was eventually excluded from the final models, noting that it should be considered as one of the most important prognostic markers.
The obtained results from the final multivariate survival models further strengthened all the findings described previously. Namely, that hepatic metastases are poor prognostic signs whether they develop synchronously or metachronously. Higher total WBC, NLR, GLR, or CRP values are associated with shorter survival times, whereas a higher mEHT treatment cycle count has the most benefit if most of the treatment sessions are completed within the first 6 months after treatment initiation. However, after the treatments are completed, other effects of progressive tumor disease almost negate the positive effects of the number of treatments, and survival from the date of the last treatment was no longer affected by the number of treatments (Table 3 and Table 4).

3.2.5. Longitudinal Survival Analysis

To investigate whether the changes in the immune marker levels were consistent with patient survival, they were included in the survival models as longitudinal, time-dependent covariates as well. It was found that the constant increase in the CRP level (HR: 1.0711; 95% CI: 1.0390–1.1040; p < 0.0001), total WBC (HR: 1.0144; 95% CI: 1.0090–1.0190; p < 0.0001), neutrophil (HR: 1.0853; 95% CI: 1.0460–1.1270; p < 0.0001) and total granulocyte count (HR: 1.0860; 95% CI: 1.0500–1.1230; p < 0.0001), and in the NLR (HR: 1.1170; 95% CI: 1.0760–1.1590; p < 0.0001) and GLR values (HR: 1.1130; 95% CI: 1.0770–1.1500; p < 0.0001) is associated with shorter survival times.

3.3. Comparison of High and Low Immune Marker Groups

To obtain further data on what might underlie the observation that only the total WBC, NLR, GLR, and CRP values of the second follow-up measurement had a high prognostic value after the mEHT treatment was completed, we created “high” and “low” sub-cohorts. As a technical step, ROC analysis and Youden’s J statistic were used to define the optimal cut-off values. The grouping variable on which the analysis was based was the survival time of less than (poor prognosis) or more than (good prognosis) 1 year, as already shown in Table 1. Based on the ROC results, the optimal cut-off value for total WBC, NLR, GLR, and CRP was 8.65 × 109/L, 7.78, 3.30, and 6.20 mg/L, respectively. The test quality of the models (area under the curve) was in the good range for all measures (total WBC: 0.7340; NLR: 0.6645; GLR: 0.6723; CRP: 0.7998). The following numbers of patients were observed in each sub-cohort: 30 low WBC and 41 high WBC, 46 low NLR and 15 high NLR, 31 low GLR and 40 high GLR, and 19 low CRP and 28 high CRP. The lower numbers of cases were due to the fact that not all patients had a second follow-up visit, as discussed in the methods section.
As an expected result, higher CRP levels were found in high-NLR (112.67 ± 87.73 mg/L vs. 22.43 ± 40.25 mg/L; p = 0.0031) and high-GLR (71.05 ± 75.81 mg/L vs. 19.86 ± 39.02 mg/L; p = 0.0002) sub-cohorts, but not in the high-WBC sub-cohort (p = 0.1732). Similarly, the neutrophil (p = 0.0096) and total granulocyte percentages (p = 0.0419), and NLR (p = 0.0054) and GLR (p = 0.0120), were significantly higher, while lymphocyte percentages (p = 0.0120) were significantly lower in the high-CRP sub-cohort. Except for the high-NLR sub-cohort, where a significantly higher number of synchronous hepatic metastases occurred (30.43% vs. 66.67%; p = 0.0417), no differences could be justified in the case of patients’ age, sex, metastasis, tumor location, and the presence of ascites. A statistically significant difference was observed in the survival of patients under and above the cut-off values in the case of all comparisons (Figure 3). Regarding the mEHT-related measures, the following difference was observed. Although the mEHT treatment cycle count was higher in every comparison (low NLR: +13, p = 0.0890; low GLR: +7, p = 0.2146; low CRP: +10, p = 0.1080), a significant difference was only observed in the high-WBC sub-cohort (low WBC: 30.95 ± 20.02; high WBC: 44.23 ± 24.32; p = 0.0250).

4. Discussion

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a usual median survival of less than 12 months, largely due to its aggressiveness and resistance to conventional therapies [8,31]. Despite the increasing incidence, effective treatment options remain limited [4]. Modulated electro-hyperthermia (mEHT) shows promise, particularly for its potential immunological benefits. However, its effects on standard immunological markers have not been thoroughly investigated. This retrospective cohort study examines the longitudinal effects of mEHT on leukocyte counts and C-reactive protein levels in patients with advanced PDAC.
An increasing body of evidence in the literature has demonstrated the beneficial effects of mEHT on various cancer patients [14,18], particularly through its impact on their immune system. E.g., in non-small-cell lung cancer patients, after mEHT treatment, a significant reduction in CRP levels and improved quality of life have been observed [19]. Regardless of additional low-dose immunotherapy and/or concomitant immunomodulatory treatment(s), mEHT is a more significant effector of longer patient survival compared to the other additional treatment options [32]. Higher and lower CD4+/CD8+ T-cell ratios can be used as an indirect marker of the immune functions’ activation and decline, respectively [33]. In hepatocellular [20] and prostate cancer [33] patients undergoing conventional hyperthermia, a change in the CD4+/CD8+ T-cell ratio was observed; however, the observation of the two studies was inconclusive, as they reported changes in opposite directions [20,34]. Combining local hyperthermia with the intraperitoneal perfusion of cytokine-induced killer cells led to enhanced cellular immunity and prolonged survival in hepatocellular cancer patients [21]. Of note, a trial with 210 ovarian cancer patients showed that mEHT combined with ionizing radiation exerts an immune-related anti-tumor effect [23]. Furthermore, evidence from another study suggests that integrating mEHT with immunotherapy may enhance survival outcomes in glioblastoma multiforme patients [22]. In the current study, the median patient survival following the first mEHT treatment was 8.15 months, including a 4-month period after the last mEHT session. Regardless of the length of the mEHT treatment, all immune biomarkers measured at the midpoint of the mEHT treatments did not differ from baseline measurements. However, following the mEHT treatment, a significant increase in the total WBC count, neutrophil granulocyte count, total granulocyte count, monocyte count, CRP level, and NLR and GLR values were observed compared to that of the baseline measurements. These observations are consistent with those observed in the case of conventional chemotherapies [35,36,37,38,39]. Elevated NLR (and GLR) values are known to be good prognostic indicators of higher tumor load, and their increase is an indication of pro-tumorigenic changes, while their decrease to normal values is considered as a good prognostic sign [40]. It is worth noting that the higher NLR and GLR values we observed after the mEHT treatment were not related to a decreased number of lymphocytes, which suggests that, with high probability, paraneoplastic hyperleukocytosis might be the cause of this observation [41,42]. Importantly, the impact of the elevated values of the aforementioned factors on the survival of pancreatic cancer patients was also confirmed by longitudinal survival analyses, which further strengthens the previously discussed mechanisms.
Several results of our study have been described in previous observations in the literature. For example, it was found that patients with tumors in the tail of the pancreas or those with hepatic metastasis were found to face a higher risk of early death, of which both are consistent with previous findings [43,44]. Interestingly, female patients tended to have longer survival, which may be related to a lower incidence of hepatic metastases and more tumors located in the head of the pancreas compared to males. In the literature, similar observations have been reported to the latter; however, most of the available data are inconclusive [35,36,38]. Although the results presented here are consistent with trends observed in the existing PDAC literature, it is important to emphasize that these findings are novel for mEHT-treated PDAC patients. Additionally, this study includes a longitudinal survival analysis, a feature that has been rarely published to date.
A further goal of our study was to increase our knowledge about the relationship of mEHT and PDAC, which has only been investigated by a few research groups worldwide. Previous studies have concluded that concomitant mEHT improves progression-free and overall survival and results in better disease response [45,46,47,48,49,50,51]. From previous studies, it is known that both metastases and ascites can significantly decrease the efficacy of mEHT [48,51], which was also observed in the current research. Moreover, Fiorentini et al. proposed that the time elapsed between the diagnosis and the first treatment and the survival calculated from the diagnosis or the first treatment are strongly associated [45]. In line with this hypothesis, the earlier initialization [50] of mEHT and a larger number of treatment cycles are associated with prolonged survival in PDAC patients [50,51]. In the current study, we were able to extend our knowledge with the observation that the higher the number of mEHT treatment cycles in the first six months after starting the treatment, the better the survival of the patients. However, once the treatments were completed, the progression of the tumor seemed to negate the positive impact of the number of treatments, rendering the survival data from the date of the last treatment unaffected by the treatment count.
Based on all of the data we observed during our study, we postulate the following hypothesis. With a high probability, after a certain point, the negative effects of the progressive tumor disease can no longer be counteracted by mEHT treatment, which is associated with an increase in immune-related markers. Due to the latter, the increase in several immunomarkers observed in this study may serve as prognostic markers for tumor progression, ultimately indicating that mEHT treatment is no longer a viable therapeutic option in those specific cases.

Limitations of the Study

A significant limitation of this study is its retrospective design, which introduces potential selection bias, incomplete data, and limited control over confounding factors. Additionally, the patient cohort was relatively small and heterogenous, and all patients had advanced disease. Furthermore, it remains unclear which variable(s) contributed to the significant elevation in neutrophil granulocyte count and CRP levels observed in the final measurement. Moreover, the data of this study originated from a single center only, and no pathological analyses on molecular and/or immunological changes could be performed to further investigate how the tumor microenvironment affected the observed results.

5. Conclusions

In conclusion, our findings suggest a significant role of the investigated immunological markers in the survival of advanced- and late-stage pancreatic cancer patients receiving concomitant mEHT treatment. Despite the limitations, the insights gained provide a solid foundation for future research. Further studies are needed to explore more specific immunological patterns corresponding to the mEHT treatment. Ultimately, our work contributes to a deeper understanding of the topic and highlights the importance of certain immunological markers.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/immuno4040025/s1, Figure S1: Change in immune-related markers at the three measurements, investigated by Wilcoxon signed-rank tests; Figure S2: The overall survival of the patient cohort; Table S1: The mean ± standard deviation of the investigated parameters at the three time points, and the Holm-adjusted p-values of the Wilcoxon signed-rank tests.

Author Contributions

Conceptualization, M.H., A.M.S. and Z.H.; methodology, N.K.D., T.G., M.H., A.S. and G.B.; formal analysis, N.K.D. and Z.H.; investigation, N.K.D., T.G., M.H., A.S., V.M.-D., J.G. and G.B.; resources, M.D. and A.M.S.; data curation, N.K.D., M.H. and Z.H.; writing—original draft preparation, N.K.D. and Z.H.; writing—review and editing, T.G., M.H., A.S., V.M.-D., G.B., J.G., M.D. and A.M.S.; visualization, N.K.D. and Z.H.; supervision, T.G., M.D., A.M.S. and Z.H.; project administration, M.D. and A.M.S.; funding acquisition, M.D. and A.M.S. All authors have read and agreed to the published version of the manuscript.

Funding

Research was supported by the National Research, Development and Innovation Office of Hungary (grant number NVKP_16-1-2016-0042). The funding bodies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Regional and Institutional Committee of Science and Research Ethics, Semmelweis University (SE TUKEB 8/2017 and SE TUKEB 8-1/2017; approval date of the latest version: 9 January 2023).

Informed Consent Statement

Informed consent was obtained from all mEHT-treated subjects involved in the study.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Acknowledgments

The authors are grateful for the help of Erika Borbenyi M.D. and Marianna Kvasnika, without whose help the study would not have been possible.

Conflicts of Interest

The authors declare no conflicts of interest.

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Immuno 04 00025 g001
Figure 1. The predicted mean ± standard error and p-values of the (A) total white blood cell count, (B) C-reactive protein level, (C) granulocyte–lymphocyte ratio, and (D) neutrophil–lymphocyte ratio at the three different study visits, obtained from the linear mixed-effects models.
Figure 1. The predicted mean ± standard error and p-values of the (A) total white blood cell count, (B) C-reactive protein level, (C) granulocyte–lymphocyte ratio, and (D) neutrophil–lymphocyte ratio at the three different study visits, obtained from the linear mixed-effects models.
Immuno 04 00025 g001
Immuno 04 00025 g002
Figure 2. The cumulative leukocyte cell counts at the baseline measurement and the two follow-ups: (A) all five leukocyte subtypes including lymphocytes, monocytes, basophil granulocytes, eosinophil granulocytes, and neutrophil granulocytes; (B) only the lymphocytes, monocytes, and granulocytes.
Figure 2. The cumulative leukocyte cell counts at the baseline measurement and the two follow-ups: (A) all five leukocyte subtypes including lymphocytes, monocytes, basophil granulocytes, eosinophil granulocytes, and neutrophil granulocytes; (B) only the lymphocytes, monocytes, and granulocytes.
Immuno 04 00025 g002
Immuno 04 00025 g003
Figure 3. Differences in the overall survival of patients with (A) total white blood cell counts (WBC) lower or higher than 8.65 × 109/L, (B) C-reactive protein (CRP) levels lower or higher than 6.20 mg/L, (C) granulocyte–lymphocyte rate (GLR) values lower or higher than 3.30, and (D) neutrophil–lymphocyte rate (NLR) values lower or higher than 7.76.
Figure 3. Differences in the overall survival of patients with (A) total white blood cell counts (WBC) lower or higher than 8.65 × 109/L, (B) C-reactive protein (CRP) levels lower or higher than 6.20 mg/L, (C) granulocyte–lymphocyte rate (GLR) values lower or higher than 3.30, and (D) neutrophil–lymphocyte rate (NLR) values lower or higher than 7.76.
Immuno 04 00025 g003
Table 1. Baseline clinicopathological characteristics of the participants of the study.
Table 1. Baseline clinicopathological characteristics of the participants of the study.
Clinicopathological CharacteristicsMean ± SD, HR + 95%CI, or
Number of Observations (Percentage)
Age at the first mEHT treatment64.58 ± 9.85
Male:female ratio35:38 (47.95%:52.05%)
Location of the tumor 1
 - Head of the pancreas41 (56.16%)
 - Tail of the pancreas10 (13.70%)
 - Body of the pancreas21 (28.77%)
Metastases
 - Synchronous hepatic27 (36.99%)
 - Metachronous hepatic12 (16.44%)
 - Synchronous peritoneal20 (27.40%)
 - Metachronous peritoneal13 (17.81%)
 - Synchronous pulmonary5 (6.85%)
 - Metachronous pulmonary8 (10.96%)
 - Other synchronous6 (8.22%)
 - Other metachronous6 (8.22%)
Ascites26 (35.62%)
Average number of mEHT treatment cycles the patients received35.84 ± 22.94
Time elapsed between tumor diagnosis and the first mEHT treatment (months)4.40 ± 5.70
Time elapsed between the first and last mEHT treatments (months)4.52 ± 3.09
Median overall survival calculated from the diagnosis of the tumor (months)13.24 (95% CI: 11.33–17.31)
Prognosis
 - Good (OS from diagnosis > 12 months)41 (56.16%)
 - Poor (OS from diagnosis < 12 months)32 (43.84%)
Median overall survival calculated from the first mEHT treatment (months)8.15 (95% CI: 7.16–10.97)
Median overall survival calculated from the last mEHT treatment (months)4.01 (95% CI: 2.73–5.72)
1 A single patient’s tumor location data were not available. CI: confidence interval, HR: hazard ratio, mEHT: modulated electro-hyperthermia; SD: standard deviation.
Table 2. The p-values of the pairwise comparisons of the investigated parameters.
Table 2. The p-values of the pairwise comparisons of the investigated parameters.
ParameterBaseline vs.
First Follow-Up		Baseline vs.
Second Follow-Up		First Follow-Up vs.
Second Follow-Up
Total white blood cell count0.68770.01870.0498
Neutrophil granulocyte count0.48470.03480.1541
Eosinophil granulocyte count0.32890.28160.9186
Basophil granulocyte count0.31430.45510.7936
Total granulocyte count0.80550.03820.0668
Monocyte count0.88860.25580.3183
Lymphocyte count0.50490.99620.5049
C-reactive protein0.62930.00030.0001
Granulocyte–lymphocyte ratio0.73190.00130.0004
Neutrophil–lymphocyte ratio0.97770.00100.0011
Table 3. p-Values obtained for the multivariate models investigating the survival time after the first modulated electro-hyperthermia (mEHT) treatment. It has to be noted that, in order to ensure model assumptions, some parameters had to be adjusted [28], for which the total observation period was split into two periods: 0–4 months and longer than 4 months. For these, two p-values were given: the first for the initial and the second for the later period.
Table 3. p-Values obtained for the multivariate models investigating the survival time after the first modulated electro-hyperthermia (mEHT) treatment. It has to be noted that, in order to ensure model assumptions, some parameters had to be adjusted [28], for which the total observation period was split into two periods: 0–4 months and longer than 4 months. For these, two p-values were given: the first for the initial and the second for the later period.
ParameterModel no.1Model no.2Model no.3
Total white blood cell count0.1033
0.0066		––
Granulocyte–lymphocyte ratio–0.1696
0.0362		–
Neutrophil–lymphocyte ratio––0.0843
0.0385
C-reactive protein0.0228
0.0113		0.2273
0.0170		0.5662
0.5460
Number of mEHT treatment cycles0.0444
0.1320		0.0242
0.0268		0.0112
0.0497
Hepatic metastases:
 - None (ref.) vs. metachronous0.01020.01890.0283
 - None (ref.) vs. synchronous0.00860.01010.0184
 - Metachronous (ref.) vs. synchronous0.68320.82770.8689
Tumor location:
 - Tail (ref.) vs. head0.07680.17720.1732
 - Tail (ref.) vs. body0.44590.71320.6154
 - Head (ref.) vs. body0.07810.08060.1338
ref.: reference category; –: the parameter was not included in the model.
Table 4. p-Values obtained for the multivariate models investigating the survival time after the last modulated electro-hyperthermia (mEHT) treatment. It has to be noted that, in order to ensure model assumptions, some parameters had to be adjusted [28], for which the total observation period was split into two periods: 0–4 months and longer than 4 months. For these, two p-values were given: the first for the initial and the second for the later period.
Table 4. p-Values obtained for the multivariate models investigating the survival time after the last modulated electro-hyperthermia (mEHT) treatment. It has to be noted that, in order to ensure model assumptions, some parameters had to be adjusted [28], for which the total observation period was split into two periods: 0–4 months and longer than 4 months. For these, two p-values were given: the first for the initial and the second for the later period.
ParameterModel no.1Model no.2Model no.3
Total white blood cell count0.0129
0.1190		––
Granulocyte–lymphocyte ratio–0.1613
0.0534		–
Neutrophil–lymphocyte ratio––0.0851
0.0342
C-reactive protein0.0021
0.0224		0.06780.2664
Number of mEHT treatment cycles0.22260.72020.8989
Hepatic metastases:
 - None (ref.) vs. metachronous0.11950.23610.1846
 - None (ref.) vs. synchronous0.05310.03210.0642
 - Metachronous (ref.) vs. synchronous0.85180.63090.8943
Tumor location:
 - Tail (ref.) vs. head0.00130.01400.0117
 - Tail (ref.) vs. body0.01290.06660.0556
 - Head (ref.) vs. body0.36840.25660.2689
ref.: reference category; –: the parameter was not included in the model.
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Dobos, N.K.; Garay, T.; Herold, M.; Simon, A.; Madar-Dank, V.; Balka, G.; Gajdacsi, J.; Dank, M.; Szasz, A.M.; Herold, Z. Immune Marker and C-Reactive Protein Dynamics and Their Prognostic Implications in Modulated Electro-Hyperthermia Treatment in Advanced Pancreatic Cancer: A Retrospective Analysis. Immuno 2024, 4, 385-399. https://doi.org/10.3390/immuno4040025

AMA Style

Dobos NK, Garay T, Herold M, Simon A, Madar-Dank V, Balka G, Gajdacsi J, Dank M, Szasz AM, Herold Z. Immune Marker and C-Reactive Protein Dynamics and Their Prognostic Implications in Modulated Electro-Hyperthermia Treatment in Advanced Pancreatic Cancer: A Retrospective Analysis. Immuno. 2024; 4(4):385-399. https://doi.org/10.3390/immuno4040025

Chicago/Turabian Style

Dobos, Nikolett Kitti, Tamas Garay, Magdolna Herold, Alexandra Simon, Viktor Madar-Dank, Gyula Balka, Jozsef Gajdacsi, Magdolna Dank, Attila Marcell Szasz, and Zoltan Herold. 2024. "Immune Marker and C-Reactive Protein Dynamics and Their Prognostic Implications in Modulated Electro-Hyperthermia Treatment in Advanced Pancreatic Cancer: A Retrospective Analysis" Immuno 4, no. 4: 385-399. https://doi.org/10.3390/immuno4040025

APA Style

Dobos, N. K., Garay, T., Herold, M., Simon, A., Madar-Dank, V., Balka, G., Gajdacsi, J., Dank, M., Szasz, A. M., & Herold, Z. (2024). Immune Marker and C-Reactive Protein Dynamics and Their Prognostic Implications in Modulated Electro-Hyperthermia Treatment in Advanced Pancreatic Cancer: A Retrospective Analysis. Immuno, 4(4), 385-399. https://doi.org/10.3390/immuno4040025

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