Upregulation of p16INK4A in Peripheral White Blood Cells as a Novel Screening Marker for Colorectal Carcinoma

Objective: Screening of colorectal cancer (CRC) is important for the early detection. CRC is relating to aging and immuno-senescence. One such senescent marker is p16INK4A expression in immune cells. The objective of the study is to investigate the protein expression of p16INK4A in peripheral white blood cells as a screening marker for colorectal cancer. Methods: A case-control studies were conducted. Cases were patients with colorectal cancer and controls were matched with cases based on age and sex. Peripheral blood was collected from patients and controls and the protein p16INK4A was measured with immunofluorescent techniques. The p16INK4A levels from cases and controls were evaluated using ROC analysis to be used as a screening marker in CRC patients. Mean fluorescent intensity of p16INK4A of cases and controls were analyzed in CD45+, CD3+ or CD14+ cells. The p16INK4A levels of cases were also correlated with clinical data. Result: Statistically significant increased expression of p16INK4A levels were found in cases compared to controls. p16INK4A in peripheral immune cells had 78% sensitivity and 71% specificity which can possibly be used as a diagnosis tool for colorectal cancer. P16INK4A-positive cell percentage and mean florescent intensity were significantly higher in CD45+ cells, CD3 positive cells and CD14 positive cells. No significant correlation was observed with the clinical data and p16INK4A level of CRC patients. Conclusion: The significant increase of p16 INK4A expression level in peripheral immune cells represents potential for use as a CRC screening marker.


Introduction
, epigenetic alterations and genomic instability (Mutirangura, 2019). The other important thing is the collective changes of the immune system, termed as immuno-senescence, can also be seen with age (Xu et al., 2020). Although immune responses may inhibit cancer growth, they can also stimulate cancer growth and metastasis, as a consequence of immuno-senescence (Pawelec, 2017) and tumor-associated inflammation (Grizzi et al., 2013). One of the aging markers is p16 INK4A .
p16 INK4A is a cell cycle regulator, functioning as a cyclin-dependent kinase inhibitor of the INK4 family (Laphanuwat and Jirawatnotai, 2019) and tumor suppressor protein (Buj and Aird, 2019). It in its canonical pathway (RB pathway) can act as an aging marker (LaPak and Burd, 2014). Increased expression of p16 INK4A is observed in pathophysiological conditions such as tumorigenesis and senescence (Liu et al., 2009).
Therefore, it is worth to elucidate p16 INK4A in CRC patients' immune cells as a marker. This research aimed to measure the protein expression of p16 INK4A in peripheral white blood cell of CRC patients for application as a diagnostic tool. Then, we investigated the CD3+ cells represented T lymphocytes and CD14+ cells represented monocytes to find whether or not they manifest the p16 INK4A protein.

Study design and target population
Cross-sectional case-control studies were done at the King Chulalongkorn Memorial Hospital. All samples were collected from May 2021 to December 2021. Normal samples were collected from patients without a family history of cancer, autoimmune diseases and showed negative CRC screening results from colonoscopy. This group served as healthy controls for this study. Sample size was calculated from the pilot study using the formula N = 2(Zα/2 + Zβ) 2 * Ϭ 2 / (X ̅ 1 -X ̅ 2 ) 2 with α significance level at 0.05 and power β of 90%, which required at least 53 participants in each group. Colorectal cancer staging was assessed by the American Joint Committee on Cancer TNM system by a pathologist. Venous whole blood (2 mL) with anticoagulant EDTA was collected from participant based on WHO guidelines.

Buffy coat layer preparation
Whole blood was centrifuged at 1000g for 12 min at room temperature. The buffy coat layer was collected, and the red blood cells (RBCs) were lysed with 20 volumes of RBC lysis solution (Abcam, USA). The lysis reaction was terminated by adding an equal volume of phosphate buffered saline (PBS) and centrifuged for 5 min at 400g. Cells were washed with PBS 3 times and white blood cells (WBCs) were counted with a counting chamber. WBCs were then fixed with 4% paraformaldehyde for 15 min (1 million cells/mL fixative), and washed 3 times with deionized water. WBCs were placed in 96-well plates (approximately 40,000 cells/ well) and stored at room temperature before immuno-fluorescence staining.

Immunofluorescence staining
Sample wells containing WBCs were washed with PBS 3 times. One hundred microliter of 0.5% Triton X-100 was added into each well and incubated for 10 min at room temperature. Wells were then washed with PBS 5 times. Then, 100μl of 5% (w/v) BSA was added into each well for 30 min. One hundred μL of diluted primary antibodies (anti-p16 INK4A and anti-CD45) in 1% (w/v) BSA, were added into each well and incubated overnight (16 h) at 4°C. Dilutions of anti-p16 INK4A primary rabbit antibody (Cell Signaling) and anti-CD45 primary mouse antibody (Abcam) were 1:1,000. Anti-CD3 primary mouse antibody (Cell signaling) and anti-CD14 primary mouse antibody (Cell signaling) were diluted 1:500. Wells were washed with 100 μL of 0.1% Tween PBS 3 times. One hundred μL of 1:1,000 diluted secondary fluorescent antibodies (anti-rabbit FITC and anti-mouse Cy3) (cell signaling) in 1% (w/v) BSA, were added into each well and incubated for 2 h at room temperature in the dark. Wells were washed with PBS 3 times. One hundred μL of DAPI (final concentration of 1μg/ml) (for nuclear staining) was added into each well for 10 min and washed with PBS for 3 times.

Image analysis
Three-color images were captured as follows: blue (DAPI 358 -461 nm); green (FITC 500 -520 nm) and red (Cy3 560 -570 nm) with a motorized fluorescence microscope IX83 (Olympus Co., Ltd., USA). All images were acquired using a defined experimental protocol throughout the study. Briefly, 20 fields of 20X objective (5 columns and 5 rows) were taken with the fixed exposure time for DAPI (7s), FITC (200ms) and Cy3 (300ms). The fluorescence intensity of cell was calculated using CellSens imaging software after setting the range of intensity values (70 to 150) and perimeter values (18 to 55 nm). Mean fluorescence intensity (MFI) was then acquired from the average fluorescence intensity of cells from the whole image. In double immunofluorescence staining, the positive region was identified by using CancerScreen.exe program (Puttipanyalears et al., 2021). P16 INK4A -positive/ CD-positive cells were identified by the signals of red and green in the same spot with the criteria of intensity value (60 to 150) and perimeter value (18-100 nm).

Ethical approval
This study was conducted under the regulations of the Institutional Review Board, Faculty of Medicine, Chulalongkorn University, COA number: 1580/2021. This study was conducted in accordance with the Declaration of Helsinki. All study participants provided informed consent.

Statistical analysis
Normal distribution values of the study groups were assessed by Shapiro-wilk test. To detect the significant differences of measurements between CRC and healthy study participants was 31 years and maximum age was 89 years. The values in samples were normally distributed according to Shapiro-Wilk test. Therefore, we calculated the difference of the two groups with the independent sample t-test. Among the total number of CRC 72 and control 79, there was also a statistically significant increase MFI of p16 INK4A in WBC of CRC patients compared to controls ( Figure 2C) (95% CI = 4.5 to 8.3, p<0.001).

Evaluation of p16 INK4A as a marker in CRC patients
p16 INK4A levels in peripheral immune cells were observed to be used as a marker in CRC patients, as the p16 INK4A level of peripheral white blood cells increased in CRC patients compared to controls. ROC curve was calculated using MFI of p16 INK4A in CD45+ cells of patients and controls. Area under the curve was 78% (p <0.001) with the standard error value of 0.04. There was 78% sensitivity and 71% specificity with Youden index of 0.48, cut-off value of 83.26 MFI (Figure 3). The 2x2 table with the numbers of true positive (TP), false negative (FN), false positive (FP) and true negative (TN) was shown in Table 2. From 2x2 table, sensitivity (TP/ TP+FN) is 78%, specificity (TN/FP+TN) is 71%, positive predictive value (TP/TP+FP) is 71%, negative predictive value (TN/FN+TN) is 78% and accuracy (TP+TN / TP+FP+FN+TN) is 74%.
control participants, independent sample t-tests was used. For comparison between more than two groups, ANOVA was calculated. Data are expressed as mean±SD, 95% CI and p-value. Receiver operating characteristic (ROC) curve and Youden index were used to evaluate the sensitivity and specificity of the marker. GraphPad prism-8 (GraphPad Software Inc., USA) was used for statistical analysis and graphical illustrations.

p16 INK4A protein level in white blood cells of cases and controls
p16 INK4A levels were measured in the peripheral white blood cells of CRC patients ( Figure 2A) and healthy controls. Since p16 INK4A levels can depend on donor age, the healthy control was age-and sex-matched to the CRC cohort. Comparison of cases with controls revealed a statistically significant increase in MFI per cell between CRC patients compared to controls ( Figure  2B) (95% CI = 2.8 to 11.9, p = 0.003).
Then, we increased the number of samples both in CRC and control, not adjusted by age and sex. The total number of CRC patients in this study was 72 with mean age of 64.54±11.3 years, while that of healthy controls was 79 with a mean age of 63.91±8.8 years. Cases and controls demographics are shown in   Table 1.

Expression of p16 INK4A in peripheral blood CD45+ cells, CD3+ cells and CD14+ cells of cases and controls
We used double immunofluorescence staining to investigate lymphocytes and monocytes positive for p16 INK4A . Antibodies of p16 INK4A and CD3 were used to detect p16 INK4A -positive lymphocytes ( Figure 5A). For monocytes population, antibodies of p16 INK4A and CD14 were used ( Figure 5B). Mean fluorescence intensity of p16 INK4A in CD3+ cells and CD14+ cells were calculated in CRC patients and healthy controls. A significant increase in levels of p16 INK4A were observed in CD3+ cells (95% CI = 0.2 to 9.1, p = 0.04) and CD14+ cells (95% CI = 0. to 6.3, p = 0.04) of CRC patients. Expression of p16 INK4A was higher in CD3+ cells than in CD14+ cells (95% CI = 0.4 to 6.6, p = 0.03) ( Figure 5C). The number of p16 INK4A -positive white blood cells increased in CRC patients, revealing a significant difference in CD45+ subset (95% CI = 3.2 to 24.3, p = 0.01), in CD3+ subset (95% CI = 0.06 to 17.1, p = 0.05) and in CD14+ subset (95% CI = 1.0 to 13.2, p = 0.02) ( Figure 6A). When the number of p16 INK4A positive cells and MFI of each cell subset were combined in a graph, there were increasing trend of higher value in p16 INK4A and percent positive cells in CRC groups ( Figure  6B, 6C and 6D).

Discussion
With immunofluorescence technique, we can measure the intensity of the positive cells and number of positive cells. The MFI of p16 INK4A in WBC of CRC patients had a significantly higher value than that of healthy control group in this study. P16 INK4A in peripheral immune cells represents 78% sensitivity and 71% specificity for application as a potential colorectal cancer screening marker. Proteins, DNA, RNA and metabolites from tissue, blood, stool and urine have been used in CRC for screening, diagnosis and monitoring, but have varying degree of success to be used as an effective biomarker (Loktionov, 2020). Colonoscopy has been used as the gold standard for the diagnosis of CRC (Hazewinkel and Dekker, 2011). Blood-based protein biomarkers for detection of CRC have various sensitivity and specificity, while using protein panel increase the detection rate of CRC (Loktionov, 2020).
The increased expressions of p16 INK4A have been notified in the peripheral blood cells of testicular cancer survivors (Bourlon et al., 2020) and breast cancer survivors (Sanoff et al., 2014). Presence of p16 INK4A in the immune cells can represent immuno-senescence (Liu et al., 2009). Immuno-senescence is initiated earlier in men than in women, likely due to hormonal differences between males and females, as estrogen enhances immune responses, while progesterone and androgens favor immune suppressive actions (Ostan et al., 2016). Therefore, we investigated age and sex-adjusted CRC patients and controls, the MFI in CRC group is significantly higher, meaning that immuno-senescence can contribute to CRC patients regardless of age and sex. Immuno-senescence can result from oxidative stress, cellular and DNA damage, chronic inflammation and cytotoxic therapy (De Padova et al., 2021). According to Giunco et al., (2019) immuno-senescence in CRC patients can lead to negative consequences like disease relapse, progression and death.
CRC group showed increased p16 INK4A levels compared to healthy controls (mean age 63.91±8.8 yrs). However, stages of CRC were not correlated to the level of p16 INK4A which is similar to the finding of Milde-Langosch et al., (2001) that reported p16 INK4A expression was not correlated with the clinical stages of breast cancer. MFI value of p16 INK4A were higher in some categories and was found to be relatively high in the categories of late stage, male patients and old age, but there were no statistically significant differences. The immune landscape in elderly population and metastasis patients differ from other patients in both innate and adaptive immune system (Weng, 2006;Fulop et al., 2017;Blomberg et al., 2018). The various factors can influence the level of p16 INK4A in immune cells, as p16 INK4A has an impact on immune surveillance (Sznurkowski et al., 2017;Leon et al., 2021). Limitations of this study include the small sample size (n) of some categories and due to the nature of this cross-sectional study, we could not prove that p16 INK4A level correlates to the prognosis of the patients.
Protein expression of p16 INK4A was higher in T cell subsets of CRC patients. T cells are important for tumor immunity and immunotherapy (Woolaver et al., 2021). Senescent T cells relate to progression of cancers (Vicente et al., 2016). Senescent T cells in CRC patients also relate to patient negative outcomes such as disease relapse, disease progression and death (Giunco et al., 2019). p16 INK4A expression in peripheral blood T cells is associated with chronologic age, molecular age, and IL-6 production (Liu et al., 2009;Burd et al., 2020). IL-6 is important in human frailty (Soysal et al., 2016) and relates to cellular senescence (Kojima et al., 2013). p16 INK4A protein expression increase in T cells of peripheral blood and bone marrow of acute lymphoblastic leukemia and its expression correlates to senescent features (Chebel et al., 2007).
In conclusion, the well-known aging marker p16 INK4A in peripheral blood of CRC patients has been observed in this study. The statistically significant increase of p16 INK4A level in WBCs revealed a potential diagnostic tool for CRC. However, the protein expressions of p16 INK4A

Author Contribution Statement
The experiments were conducted and designed by CP and AM. The clinical samples were collected by KAT, PA and CP. KAT and CP performed and optimized the immunofluorescent staining. The results were analyzed and interpreted by KAT, CP and AM. KAT wrote the manuscript. CP, SWE and AM reviewed and edited the manuscript. All authors read and approved the final manuscript.