Lower mean corpuscular hemoglobin concentration is associated with favorable prognosis of resected pancreatic cancer.

Objective: This study was designed to explore the value of mean corpuscular hemoglobin concentration (MCHC) for overall survival (OS) in resectable pancreatic cancer (PC). Background: Systemic inflammatory response are important factors that promotes the occurrence and metastasis of malignant tumors. MCHC are suggested to be relevant to the prognosis of several malignancies. Such as lung cancer, head and neck cancers. However, there are few studies to explore the correlation between them and PC. Methods: The present study included totally 544 patients with PC who underwent radical resection between March 2011 and May 2019. These patients were classified into a training set (n=195) and a validation set (n=349). Kaplan-Meier curve method and Cox proportional hazard regression model were employed to analyze prognosis. Results: Survival curve showed that OS were significantly favorable for resectable PC patients with low preoperative MCHC (P=0.015). Multivariate analysis suggested that MCHC (hazard ratio [HR], 1.266; 95% confidential interval [CI], 0.895-1.792; P=0.028) were independent prognostic factors for resectable PC patients. Conclusion: Low MCHC was positively correlated with OS in resectable PC, which could be used to predict the prognosis.


Introduction
At present, pancreatic cancer (PC) is the most malignant tumor of the digestive system. According to the statistics, it is the third cancer-related death in the United States, and possibly turns into the second cancer-related death in the United States by 2030 [1]. The median survival time for advanced pancreatic cancer is less than 6 months, and the overall 5-year survival rate is less than 6% [1]. Surgical resection still remains the only way to cure pancreatic cancer [2]. However, the survival of patients with pancreatic cancer has not been improved significantly. Besides, 5-year survival rate is inferior to 20% [3]. Therefore, it is of great significance to find an effective prognostic index to provide personalized treatment for patients who underwent radical pancreatomy.
Inflammatory biomarkers have become the research hotspots, a large number of studies have been conducted globally. Erythrocyte-related serological markers also belong to part of inflammatory biomarkers, and erythrocyte-related inflammatory biomarkers have been shown to be associated with prognosis and recurrence in various different cancers. RDW is a hot research topic of erythrocyte-related inflammatory factors in recent years. The study found that RDW was linked to the prognosis of many malignancies, including gastric cancer [5], blood cancer [6] and kidney cancer [7]. MCHC was found to be associated with prognosis of oral cavity and lung cancer [8], especially related to the onset of prostate cancer [9]. However, few studies have explored the relationship between erythrocyte-related serological markers and pancreatic cancer. Therefore, we conducted this retrospective analysis with the aim to explore the relationship between both of them.

Patients
A retrospective review on maintained database was performed as much as possible to identify all patients underwent radical pancreatomy at Harbin Medical University Cancer Hospital from March 2011 to May 2019. Exclusion criteria were as follows: (1) patients who have not underwent radical pancreatomy, (2) patients who have not underwent routine blood examination before surgery, (3) patients with metastatic disease, (4) patients received any other treatment before enrollment (5) patients who have underwent malignant disease in other organs, and (6) patients who had incomplete/inaccurate medical records.
Patients with burns, severe vomiting, diarrhea, chronic carbon monoxide poisoning, cardiac compensatory dysfunction, and hereditary balloon disease affecting MCHC outcomes were excluded [10]. All included patients (n=544) were assigned to either a training set (n=195) or a validation set (n=349).

Data collection
Clinicopathologic information was obtained from the medical data platform of the Harbin Medical University Cancer Hospital by two independent investigators. During this procedure, the two independent investigators consulted the medical data platform to resolve any possible bias. These data included the following clinicopathologic information: age, sex, body mass index (BMI), hemoglobin (HBG), mean corpsular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), carcinoma-embryonic antigen (CEA). The following clinical information was also collected: date of surgery, tumor location, tumor size, stage, differentiation grade, nerve invasion, vascular invasion, pathological staging. The definition of resectable PC referred to the criteria defining resectability status of NCCN Clinical Practice Guidelines in Oncology (3th-edition 2019). All patients with resectable PC underwent R0 surgery and had TNM staging based on postoperative pathology. The TNM stage mentioned referred to the Joint Committee on Cancer (8th Edition).

Blood sample collection and measurements
Blood samples for routine blood examination were collected via standard venipuncture of the veins in the antecubital fossae (anterior elbow veins). Routine hematology tests, including HBG, MCH, MCHC, RDW, CEA, CA199. 2 ml EDTA anticoagulated peripheral blood and 2 ml plasma anticoagulated by sodium citrate as well as serum samples were obtained before breakfast within 7 days prior to surgery.
The samples were promptly centrifuged and processed within two hours. The cell counting of peripheral blood was measured using the SYSMEX XN-9000 full-automated hematology analyzer (Sysmex, Tokyo, Japan).

Follow-up evaluation
During a mean longitudinal follow-up period of 67 months (from a range of 22 to 103 months) after the surgery, 186 deaths events occurred (58.83%). Patients were followed every three months until death or the end of the study. The final follow-up evaluation was performed in November 2019. The follow-up data included date of primary resection, and date of death. Most follow-up includes physical examination, laboratory tests (blood routine, blood biochemistry, tumor marker CEA), and imaging studies. The primary end point OS was defined as the time from surgery to death from any cause, the latter was defined as the date of last live follow-up during this study (cut-off Nov 2019)

Statistical methods
Descriptive statistical methods were used to analyze the epidemiological, pathological, and hematological characteristics of the patients. Survival curves were drawn according to the Kaplan-Meier method, survival comparision was executed by Log-rank test. Cox proportional hazard regression model was employed for univariate and multivariate analysis. Statistically significant variables (P<0.05) found in univariate analysis were entered into the Cox regression multivariate model using the backward condition method. All tests were double-sided, and statistical significance was inferred at a P value of 0.05. All statistical analysis was accomplished using SPSS for Windows version25.

1.Clinical features of patients
Of the 544 PC patients included in the study, 195 patients were assigned to the training group and 349 patients were assigned to the validation set. The training set detailed baseline characteristics of the resectable PC patients were shown in Table 1A. The median follow-up time was 762 days. and 95 patients died during follow-up. 114(58.46%) were male. In 132(67.69%) patients, the tumor was located in the head of the pancreas. According to the TNM stage, the majority of patients 112(57.44%) were classified as stage II. The baseline level of the red blood cell correlation index in the training set is shown in Table   1A. The clinicopathological features of resectable PC patients in the validation set are shown in Table   1B. As shown in Table 1B (Table 1A). The detailed data of ROC curve analysis are shown in Table 3 and Figure 1.

3.MCHC and MCH were associated with prognosis of resectable PC
The Kaplan-Meier survival curves indicated that patients in low MCHC(p<0.001) and low MCH (p=0.026) groups had longer OS than those in the respective high-value groups ( Figure.

MCHC was an independent prognostic factor for resectable PC.
The Schoenfeld residual method was used to determine the Proportional hazards assumption, referred to as the PH assumption. The basic assumption of the PH hypothesis is: the influence of covariates on survival rate do not change with time, the hazard ratio h(t)/h0(t) is a fixed value. The results of the Cox regression model are meaningful only when the conditions of the PH assumption are met. The results showed that there was no correlation between the Schoenfeld residuals of the parameters involved in this study and the rank of failure time, which suggested that the hazard ratios of all covariates would not increase with time. It could be considered that the PH assumption was satisfied and appropriate Perform Cox regression analysis directly. The specific details were shown in Figure 4. were independent prognostic factors for resectable PC (Table 4).

DISCUSSION
The evidence indicated that tumor-associated inflammation and tumor microenvironment play a more and more important role in the cancer development, progression, metastasis [11], and clinical prognosis [12]. As reported previously, MCHC were closely related to the prognosis in several types of cancers. In the present study, it could also be observed that low MCHC can indicate better OS. Consequently, it is reasonable to combine MCHC as a simple and convenient marker so as to enrich the stratification of prognosis in PC patients.
MCHC means that the average red blood cell hemoglobin concentration is calculated by dividing the red blood cell specific volume (HCT) by the average red blood cell volume (MCV), which is an indicator of the average red blood cell volume [13]. MCHC indirectly reflects the degree of anemia from a quantitative perspective. Admittedly, anemia was associated with poor survival rates for a variety of malignancies. Qu found that low MCHC (<335) was linked to unfavorable prognosis of resected lung cancer [7]. Additionally, studies also showed that MCHC was associated with prognosis for oral and head and neck cancers. Based on the obtained findings, preoperative MCHC was an independent predictor of OS for pancreatic cancer patients undergoing curative surgical resection. To date, only a few preliminary hypotheses explain why MCHC may affect prognosis. Nevertheless, the mechanism does not remain clear. This may be related to the following mechanism. At first, Hemoglobin is the main driving force for carrying iron, and anemia can lead to excessive iron load. Iron is a trace element involved in a wide range of human metabolism. Iron deficiency and excess can lead to disease. Iron deficiency has been linked to anemia [14], chronic heart failure [15] as well as other conditions. Excess iron increases the risk of cancer [16]. Even if the excess is physiological, the risk of cancer can still increase [17]. In other words, proper iron consumption achieves a protective effect on the body. The correlation between hematological parameters and serum iron markers may be attributed to changes in iron levels that are reflected in erythrocyte morphology to some extent [18]. Second, iron overload often accelerates and induces oxidative stress. Oxidative stress is another factor that causes cancer to develop, and serum iron can induce and accelerate the current process [19]. Furthermore, the changes of red blood cell parameters indirectly reflect the process of oxidative stress. Third, Hemoglobin is the main driving force for carrying oxygen, and low hemoglobin can cause tissue hypoxia. Hypoxia can promote tumor progression and induce changes in the cell genome to enhance tumor growth and angiogenesis. Hypoxia induces epithelial mesenchyme transformation (EMT) [20], which is critical for cancer progression and metastasis. In addition, hypoxia also affects angiogenesis [21]. On the conditions of hypoxia, cancer cells exhibit adaptive metabolic changes. These include the conversion of glucose to lactic acid and increased glucose uptake through promotive glucose transporters (GLUTs), which is a phenomenon also known as the Warburg effect [22].Finally, nutrition is another factor contributing to the development of disease [23].
Reduced hemoglobin indicates that patient's nutritional status is relatively poor. There exists the increased incidence of complications and increased mortality in patients with nutritional risks before surgery [24]. In clinic, accurate pretreatment staging is essential for the treatment decision. Our study found that preoperative MCHC is an independent prognostic factor for pancreatic cancer, which can affect the prognosis of pancreatic cancer patients. Therefore, as the novel and easily obtainable prognostic marker, preoperative MCHC can be used as a supplement to pathological stages, which can also provide a more accurate prognosis.
However, the present study also had some flaws. At first, the current work was a retrospective study.
Although we had excluded factors such as blood system diseases and inflammatory diseases that might cause MCHC changes before the study, there were still a lot of unavoidable interference factors which could affect the results of this experiment. Then, this study was single-centered with small sample,