Does diabetes affect paraneoplastic thrombocytosis in colorectal cancer?

Abstract Background A large variety of factors can affect colorectal cancer (CRC) survival, including type 2 diabetes mellitus (T2DM) and paraneoplastic thrombocytosis. Although several common factors play a role in their development and platelets are damaged in both diseases, the combined relationship of the three conditions was never investigated previously. Methods A prospective, real-life observational cohort study was conducted with the inclusion of 108 CRC patients and 166 voluntary non-CRC subjects. Plasma interleukin-6 and thrombopoietin levels were measured. Results Study participants were divided into cohorts based on the presence of T2DM. Platelet count (p < 0.0500) and interleukin-6 (p < 0.0100) level were significantly higher in the CRC groups. Thrombopoietin level was higher in the T2DM, CRC, and CRC + T2DM groups (p < 0.0500). Analysis of parameter changes over time and survival models revealed that neither platelet count, interleukin-6, nor thrombopoietin levels were affected by T2DM. Death of patients was associated with higher baseline platelet count (p = 0.0042) and interleukin-6 level (p < 0.0001). Conclusion Although the independent, disease-worsening effect of paraneoplastic thrombocytosis and T2DM is known, the coexistence of the two did not further impair the survival of CRC patients, suggesting that T2DM has no significant effect over paraneoplastic thrombocytosis.


Introduction
Thrombocytosisplatelet counts above the upper value of normal range (usually ≥400 × 10 9 /L)is described as a poor prognostic sign in colorectal cancer (CRC). Both preoperative and postoperative thrombocytoses are associated with worse patient survival [1,2]. Thrombocytosis may develop for several reasons, such as the bleeding of the tumor or a metabolic change caused by the tumor itself called paraneoplastic thrombocytosis [1,3]. The proposed paracrine-signaling pathway of paraneoplastic thrombocytosis [3,4] includes the overproduction of various cytokines (e.g., interleukin-6) by the tumor, which causes increased hepatic thrombopoietin production that modulates the production of platelets within the bone marrow, ultimately resulting in an increased platelet count.
Diabetes mellitus is one of the most prevalent diseases in our time; based on the latest estimations available in the ninth edition of IDF Diabetes Atlas [5], the incidence of the disease varies between 4 and 10.4%, with over 460 million diabetes patients around the world [5]. Approximately 90% of diabetes patients are suffering from type 2 diabetes mellitus (T2DM), which develops in later ages [6], similar to that of CRC [7]. Over the age of 60 years, T2DM may occur in every fourth or fifth person [5,8], and compared to the healthy population, an increased occurrence of malignancies is confirmed in T2DM [9][10][11]. CRC occurs approximately 1.5× more often in patients with T2DM [12,13]; furthermore, the coexistence of T2DM is associated with an increased risk for a shorter survival in CRC [14,15]. Similar to CRC, T2DM can be also described by various platelet abnormalities and an increased thrombopoietin production is also known [16][17][18].
Although several potential mechanisms link cancers and T2DM together [19], and platelets are affected in both diseases [1][2][3][4][16][17][18], the relationship among T2DM, (paraneoplastic) thrombocytosis, and CRC is poorly investigated. An earlier retrospective study [20] from our group has found that a personalized thrombocytosis measure calculated from preoperative and precancer platelet counts can better predict the patient survival, and it has been associated with various clinicopathological parameters, including T2DM. Although our earlier study has revealed some possible connections between the three conditions, the exact biological effect of T2DM on CRC-related paraneoplastic thrombocytosis could not have been investigated via that retrospective manner. Therefore, a prospective, real-life observational cohort study was conducted where paraneoplastic thrombocytosis was investigated within diabetic and nondiabetic CRC cohorts through the changes in platelet counts, plasma interleukin-6, and thrombopoietin levels.

Patients and study design
A prospective, real-life observational cohort study was carried out. A total of 108 patients diagnosed with CRC and 166 voluntary non-CRC subjects were enrolled for the study between 2017 and 2019. CRC patients attended at both the Department of Internal Medicine and Hematology, Semmelweis University, Budapest and the Department of General Surgery, Szent Imre University Teaching Hospital, Budapest. Written informed consent was collected from all study participants before performing any study-specific procedures. Exclusion criteria included age <18 years, any previous malignancies, known inflammatory bowel-and/or chronic kidney-and/or systemic autoimmune-and/or inadequately controlled thyroid-and/or hematologic-and/or any mental diseases, the usage of erythropoiesis-stimulating agents and/or recent blood transfusion, and patients with an Eastern Cooperative Oncology Group (ECOG) performance status >2.
Voluntary non-CRC subjects consisted of healthy young controls, older controls, and T2DM patients. Rationale of the inclusion of healthy young subjects was that some of the measured parameters have no reference values and that the older volunteers included both completely healthy individuals and nonmetabolic disease patients (e.g., hypertension). T2DM patients attended at the Metabolic Outpatient Clinic of the Department of Internal Medicine and Hematology, Semmelweis University, Budapest. In addition to the exclusion criteria described by CRC patients above, voluntary nondiseased subjects were excluded in the presence of prediabetes or any other metabolic disorders.

Clinical and laboratory data measurements
Anamnestic data including comorbidities and recent medications, body weight, and height were collected, and fasting blood samples were drawn according to the following protocol: (1) at the diagnosis of CRC, before any oncological treatments or the surgical resection of the primary tumor, (2) at least 6 weeks, (3) 6 months, and (4) 12 months after the tumor removal surgery. At the time of postoperative measurements, oncological treatments were stopped/paused only as part of the patients' standard of oncological care, and sampling was performed only at least 6 weeks after the last treatment due to the known platelet influencing effects of several cytotoxic regimens [21,22]. If it was not feasible to stop/pause the oncological treatment, in the best interest of the patient, the visit was omitted. Complete blood count, aspartate-and alanine transaminase, gamma-glutamyl transferase, plasma glucose, and creatinine were measured at the Central Laboratory of Semmelweis University and at the Central Laboratory of Szent Imre University Teaching Hospital. Estimated glomerular filtration rate was calculated using the Chronic Kidney Disease-Epidemiology Collaboration equations [23]. Plasma interleukin-6 and thrombopoietin levels were measured using the ELECSYS ® Interleukin-6 electrochemiluminescence immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the Human Thrombopoietin Quantikine ® enzyme-linked immunosorbent assay (ELISA) (catalog number: DTP00B, R&D Systems, Minneapolis, MN, USA) kits, respectively. Plasma thrombopoietin level was measured from platelet-poor plasma, as per manufacturer description. Side of CRC was described as right sided if the tumor was originating from cecum, ascending colon, and proximal two-third of the transverse colon and left sided if originating from the distal one-third of the transverse colon, descending colon, sigmoid colon, and rectum [24]. Staging was given by histopathological examination of surgical specimens and imaging studies; the American Joint Committee on Cancer grouping was used [25]. The usage of biological agents was recorded as a dummy variable and chemotherapy was grouped as adjuvant if no metastasis and first-line, second-line, etc., if metastasis was present. The overall survival of patients was defined as the length of time from the date of CRC diagnosis until death. Follow-up of cancer patients was terminated on January 31, 2021, patients alive at this time point were right censored.

Statistical analysis
Statistical analysis was performed with R version 4.0.5 [26]. Wilcoxon-Mann-Whitney U test, Fisher's exact test, Kruskal-Wallis test with p-value corrected pairwise Wilcoxon-Mann-Whitney U tests as post-hoc, and Spearman rank correlation were used. To detect the changes of various parameters in time, linear mixed effect models were used (R-package nlme [27]). Survival of patients was analyzed for both preoperative and longitudinal data with Cox regression models and Bayesian univariate and multivariate joint survival models (R-package rstanarm [28]), respectively. p < 0.05 was considered as statistically significant, and p-values were corrected with the Holm method [29] for the problem of multiple comparisons. Compared to the "conventional" frequentist methods, Bayesian methods give only a probability distribution for the investigated parameter but no p-values. Interpretation of joint model results was as follows. If the Bayesian equivalent to the frequentist confidence interval (CI), the 95% credible interval contained the hazard ratio (HR) of 1, the model was considered as clinically not relevant. However, if the 95% credible interval was less than or more than HR = 1, the effect of the parameter was considered as a good or bad sign of patient survival, respectively. Results were expressed as mean ± standard deviation and as the number of observations (percentage) for continuous and count data, respectively. Naïve Kaplan-Meier and longitudinal survival curves were drawn with the R-package survminer (Kassambara, Kosinski, and Biecek, version 0.4.9, 2021) and the built-in methods of rstanarm, respectively.
Informed consent: Informed consent has been obtained from all individuals included in this study.

Results
A total of 274 study participants were included: 108 CRC patients and 166 voluntary, non-CRC subjects. CRC patients were divided into two cohorts based on the presence of T2DM. Patients without diabetes (n = 82) were assigned to the "CRC" group, whereas patients with a history of T2DM (n = 26) were assigned to the "CRC + T2DM" group. Duration of diabetes was 6.88 ± 6.10 years, from which T2DM was diagnosed in four cases around the time of CRC diagnosis.
In most parameters, the two tumor groups did not differ from each other, whereas plasma glucose was significantly higher (p = 0.0018), and tendentiously higher white blood cell-, neutrophil-, eosinophil-, and monocyte counts were observed in the CRC + T2DM group. Platelet aggregation inhibition therapy was more common in CRC + T2DM patients; no difference was found in radiotherapy (neoadjuvant only), whereas no-line or late-line chemotherapy was more common in those CRC patients with T2DM than those of without (p = 0.0560; Table 1).
Voluntary non-CRC subjects were divided into three cohorts: 51, 50, and 65 subjects were assigned to the "Young controls," "Control," and "T2DM" groups, respectively. Duration of diabetes was 14.91 ± 9.50 within the T2DM group. Anamnestic, clinical, and laboratory parameters of control groups are shown in Table 2. Comparison between the volunteer groups revealed that, as expected, Young controls had the lowest body mass index (p < 0.0001 vs T2DM and Controls), systolic blood pressure (p = 0.0250 vs Controls, p = 0.0410 vs T2DM), mean corpuscular volume (p = 0.0002 vs Controls, p = 0.0041 vs T2DM), red blood cell distribution width (p = 0.0023 vs Controls, p < 0.0001 vs T2DM), fasting plasma glucose (p < 0.0001 vs Controls and T2DM), and the highest heart rate (p = 0.0002 vs Controls, p = 0.0242 vs T2DM) and estimated glomerular filtration rate (p < 0.0001 vs Controls and T2DM). T2DM patients had the highest body mass index (p = 0.0098 vs Controls, p < 0.0001 vs Young controls), white blood cell count (p = 0.0430 vs Controls, p = 0.0008 vs Young controls), gamma-glutamyl transferase (p = 0.0633 vs Controls, p = 0.0003 vs Young controls) and fasting plasma glucose (p < 0.0001 vs Controls and Young controls). Significant differences of comparisons among all five groups have been indicated in Table 2.
The duration of diabetes was shorter within the CRC + T2DM group compared to those of the T2DM group. A higher proportion of oral antidiabetic drug usage and diet-only therapy was observable within the CRC + T2DM group, whereas the need for insulin therapy was greater in those within the T2DM group (p = 0.0006). Furthermore, lower glycated hemoglobin (HbA 1C ) level and fewer diabetic complications were found in the CRC + T2DM patients ( Table 3). The occurrence of hypertension (p = 0.7790) and the proportion of previous major cardiovascular event(s) before the first visit (p = 0.7004) did not differ between the two diabetic groups. Comparison of diabetes-related parameters is summarized in Table 3.

Baseline measurement of paraneoplastic thrombocytosis-related parameters
Plasma level of paraneoplastic thrombocytosis parameters of patients was compared to those of all control groups at the time of CRC diagnosis. Highest platelet counts were observed within the two tumor groups: platelet count of CRC patients was significantly higher than those of within the T2DM group (p = 0.0369), whereas the platelet count of the CRC + T2DM group was significantly higher than all of the control groups (p = 0.0369 vs Young Control and Control, p = 0.0278 vs T2DM; Figure 1a). Lowest plasma interleukin-6 levels were observed within Young controls (p < 0.0010 vs all other cohorts). Subjects of the Control and T2DM groups had similar interleukin-6 levels; and interleukin-6 was significantly higher in both tumor groups, compared to all of those observed in control groups (p < 0.0001 CRC vs Control and T2DM, p = 0.0011 CRC + T2DM vs Control, and p = 0.0069 CRC + T2DM vs T2DM; Figure 1b). Thrombopoietin level was basically the same in the Young control and Control groups, and a separate cluster was formed by the remaining three groups. The highest thrombopoietin levels were observed in the CRC + T2DM group (Figure 1c). No further difference could have been justified in any of the study groups if they were further subdivided by the usage of platelet aggregation inhibition therapy, antidiabetic drugs, or in the presence of any diabetic complications.

Changes in the parameters of paraneoplastic thrombocytosis with the course of CRC
For the 108 CRC subjects, a total of 215 measurements were available. 108, 48, 37, and 22 preoperative, postoperative, 6-month, and 12-month measurements were available, respectively. Significant decrease in later measurements occurred due to the death of patients, disease progression resulting in higher ECOG performance status and patient's unavailability to attend at later visits, the need to initiate chemotherapy earlier than the postoperative visit window, or continuous chemotherapy without drug holiday after the second study visit. Due to the decreasing number of follow-ups, a more robust statistical method not sensitive to the loss of follow-up had to be chosen; therefore, it was investigated via age-corrected and stage-corrected linear mixed effect interaction models as to how T2DM and worse clinical outcome (death) affect the changes of platelet count, interleukin-6, and thrombopoietin levels with the course of the disease. Average survival time: 16.96 ± 11.43 months, all within the first 3 years after CRC diagnosis. Diabetes had no significant effect on any of the changes (platelet count: p = 0.1190; interleukin-6: p = 0.5571; thrombopoietin: p = 0.3062, Figure 2a, c, and e). Platelet counts of all patients decreased, but among those patients who died during the time of the study, the average baseline platelet count was significantly higher (273.70 vs 353.00 × 10 9 /L; p = 0.0042) and a faster decrease could have been seen within the first 12 months after the tumor removal surgery compared to those of who survived (Figure 2b). Similarly, increased baseline interleukin-6 levels (5.76 vs 27.42 pg/mL; p < 0.0001) and marginally faster decreasing levels (p = 0.0613) were observed in deceased patients over time, whereas in those who survived the plasma interleukin-6 level was constant during our observation (p = 0.1273, Figure 2d). The initial thrombopoietin level did not differ between surviving and deceased patients (p = 0.5747), and its change was not affected by the worse clinical outcome (p = 0.5940, Figure 2f). Usage of radiotherapy and chemotherapy did not affect any of the response variables, if included within any of the models.

Survival analysis of paraneoplastic thrombocytosis-related parameters and T2DM
Thirty of the 108 patients (27.8%) died during the study, from which 20 and 10 belonged to the CRC and CRC + T2DM group, respectively. Patients were followed up no later than January 31, 2021. The survival analysis was performed on both the preoperative (single-time) and longitudinal data. Despite the higher occurrence of death (24 vs 40%) within the diabetic tumor group and the difference that appears to be significant on the naïve Kaplan-Meier figure (Figure 3), the univariate Cox model of preoperative data suggested that T2DM had no effect on patient survival (p = 0.1450   To analyze the effect of paraneoplastic thrombocytosis-related parameter changes in time, Bayesian joint models were used. First, three univariate joint models (a single parameter is analyzed within the longitudinal submodel) were constructed: the survival effect of the changes of platelet count, interleukin-6, or thrombopoietin over time was included in the longitudinal submodel, and T2DM was included in the survival submodel. An additional multivariate joint model was also constructed, where all three paraneoplastic thrombocytosis parameters were included within the longitudinal submodel, and no change was applied to the survival submodel. Interpretation of the clinical significance of parameters on patient survival was assessed as described in Section 2.
Based on the result of the univariate joint models, after the surgical removal of the primary tumor, higher platelet count and plasma interleukin-6 level is a sign of poorer survival (Figure 4). Thrombopoietin and diabetes did not affect the survival of patients in any of the univariate models. Multivariate joint model results suggested that interleukin-6 had the strongest effect on patient survival, whereas the platelet count was marginal; thrombopoietin and diabetes had no clinically relevant effect, similar to those observed in univariate models (Figures 4 and 5).

Discussion
In CRC, the occurrence of thrombocytosis, either prior or after the primary tumor removal surgery, is associated with shorter survival of patients [1,2,[30][31][32][33]. The background of thrombocytosis may vary due to several factors, including the bleeding (reactive thrombocytosis) or metabolic changes (paraneoplastic thrombocytosis) of the tumor. In the latter, the tumor produces cytokines, such as interleukin-6, in higher quantities, and those higher cytokine concentrations stimulate hepatic thrombopoietin production, which ultimately results in the overproduction of platelets [4,34]. A platelet count of >400 × 10 9 /L or >450 × 10 9 /L is the most common definition of thrombocytosis [1,3,35], but several earlier studies reported other cut-off values [1], indicating that the definition of thrombocytosis may not be perfect. In an earlier study [20], we have reported that a personalized, relative platelet measure can predict disease outcome significantly better than "traditional" thrombocytosis, which further strengthened that the definition of thrombocytosis needs to be revised. The biochemical detection of paraneoplastic thrombocytosis in CRC is a novel research area [36]. In the current study, we observed higher interleukin-6 and thrombopoietin levels of CRC patients, compared to those of control subjects, supporting the presence of paraneoplastic thrombocytosis, which was further strengthened by the result of correlation and survival analysis, in line with earlier findings [1,2,[31][32][33][36][37][38].
There is a strong relationship between CRC and various cytokines, including interleukin-6, which can play significant roles both in the development and progression of the disease [39][40][41]. Specifically, interleukin-6 is known to for its significant role in tumor proliferation, migration, induction of microsatellite instability, and angiogenesis [42,43]. It has been reported that interleukin-6 is produced by cancer-associated mesenchymal stem cells [44]. When combined with glycoprotein 130 (gp130, synonyms: IL6ST or CD130) interleukin-6 can regulate disease progression through the Shp2-Ras-ERK, JAK1/2-STAT3, and PI3K-Akt-mTOR pathways, and its higher serum levels were associated with larger tumor size, presence of metastases, and worse overall-and disease-free survival [40,41,45,46]. Due to its strong connection with CRC, interleukin-6 has been proposed as a good prognostic marker of CRC [47][48][49]. In the current study, we could also confirm the prognostic role of interleukin-6 over patient survival. The result that preoperative interleukin-6 did not affected survival was possibly due to the heterogeneity of study population, which was somewhat confirmed by the results obtained from the multivariate Cox models, as the predictive effect of interleukin-6 on survival increased. As a novel result, we found that constantly higher interleukin-6 level could have been observed in those patients, who died within the first 3 years after CRC diagnosis.
Research on CRC and thrombopoietin is limited. Earlier studies have identified that cancer cells can induce Figure 4: Longitudinal and survival predictions of the Bayesian joint models. No difference was found between CRC patients with or without T2DM. Increasing platelet count and plasma interleukin-6 level were associated with a higher risk of shorter survival times (left), whereas no change or a slow decline in these parameters is a good prognostic sign (right). Thrombopoietin levels had no clinically relevant effect neither in univariate nor in multivariate survival models.
circulating thrombopoietin production [50]; furthermore, higher thrombopoietin level is associated with gastrointestinal cancers, and a possible relationship between more advanced clinical stages and thrombopoietin has been also suggested [51,52]. Similar to the observations above, we found that the thrombopoietin level of CRC patients is higher than those of healthy subjects; however, the effect of thrombopoietin levels over survival was negligible, and no change with the course of the disease could have been identified in either of our longitudinal models.
T2DM, similarly to (paraneoplastic) thrombocytosis, has a known negative effect on CRC [15,53]. Several potential mechanisms link the two diseases together [19], including the increased plasma glucose levels (hyperglycemia), the presence of insulin resistance and hyperinsulinemia, increased insulin-like growth factor-1 levels, increased oxidative stress, higher cytokine concentrations, and increased platelet activation [9,10,13,19,53]. CRC is known to have an increased incidence in T2DM patients compared to those of within the healthy population [9,10]; a 1.3-fold increased risk of CRC has been reported [54,55]. T2DM has been described to have a negative effect on overall-, cancer-specific-, disease-free-, and recurrence-free survival of CRC patients [56,57]. In contrast to earlier findings, we found that T2DM did not affect patient survival in the current study statistically, but it has to be noted, that the percentage of patients who died was higher in the CRC + T2DM group (24 vs 40%), and similarly, the survival curves on Figure 3 also suggested a tendency toward significant difference. The effect of T2DM on patient survival was further investigated through the presence of diabetic complications, the preoperative HbA 1C level, and the duration of T2DM. Despite the fact that the duration of diabetes was basically twice as long in patients of the T2DM group, a greater number of diabetic complications were present, and a more significant proportion of patients required (intensive) insulin therapy, no significant effect could be justified for any of the above-mentioned parameters. It has to be mentioned though that the sample size of CRC + T2DM patients was low, but we could not even prove tendentious differences similar to that of observed in Figure 3.
The relationship between CRC-related paraneoplastic thrombocytosis and T2DM through biochemical measurements has not been investigated previously. It has to be mentioned though, that various platelet abnormalities and increased interleukin-6 and thrombopoietin production are known in T2DM [16][17][18][58][59][60]. Due to the abovementioned effects and the high risk of cardiovascular events, the usage of platelet aggregation inhibition therapy is very common in T2DM [61,62]. Based on the available literature, our prestudy hypothesis was that CRC patients who also suffer from T2DM would probably have higher interleukin-6 and thrombopoietin levels than those who are not affected by T2DM. In contrast to our hypothesis, no differences could have been justified in any of the parameters related to paraneoplastic thrombocytosis between the CRC and CRC + T2DM groups. Furthermore, platelet aggregation inhibition also did not have any effect on the parameters of paraneoplastic thrombocytosis even though that the therapy Figure 5: Results of the Bayesian joint survival models. Platelet count and/or plasma interleukin-6 level increasing over time is a poor prognostic factor, whereas diabetes and plasma thrombopoietin changes did not affect patient survival (Note: credible interval is the Bayesian equivalent of the frequentists' CI.).
was more common within the CRC + T2DM group. Similarly, the diabetes-related parameters also did not affect the paraneoplastic thrombocytosis-related parameters. The fact that plasma thrombopoietin level of non-CRC T2DM patients was more similar to those of CRC patients than those of control subjects was most likely related to the already known fact that thrombopoietin levels are higher in T2DM [58], but no previous data are available on how similar these values of CRC and T2DM patients should be. The presented data suggest that T2DM does not increase the effect of CRC-related paraneoplastic thrombocytosis, in most probability, and the disease-worsening effect of T2DM, which has been described in earlier publications [11], must be carried out through other factors. To identify those factors, further investigations are needed.

Limitations
Limitations of the current study were the small sample size and the heterogeneity of CRC populationthe latter may be also compensated with a larger sample size. The proportion of T2DM patients within the tumor cohort corresponded to the healthy Hungarian population (approximately every fourth person, over 60 years of age [8]). Due to patients' decision, a large number of potential subjects did not agree to be included in the study. Despite the low number of cases, it is important to emphasize that the presence of paraneoplastic thrombocytosis could have been already detected at such a low number of cases, showing its significance in CRC. The low number of T2DM in those of CRC patients allowed us only to pinpoint tendentious differences in several parameters.

Conclusion
To summarize the results of the current study, our data suggested that although some metabolic changes do occur to platelet counts and to the interleukin-6 and/or thrombopoietin synthesis in T2DM and in paraneoplastic thrombocytosis-affected CRC patients, no combined effect have been observed. Based on the current study, there is no significant relationship between the two conditions with high probability and the known disease-worsening effect of diabetes on CRC survival is presumably independent of paraneoplastic thrombocytosis.