The Pre-treatment Systemic Inflammatory Response Biomarkers areImportant Determinant of Prognosis for Patients Undergoing Neoadjuvant Therapy for Rectal Cancer

Purpose: To evaluate the prognostic potential of inflammatory response biomarkers neutrophil to lymphocyte ratio (NLR), derived neutrophil to lymphocyte ratio (dNLR), platelet to lymphocyte ratio (PLR) and lymphocyte to monocyte ratio (LMR) in predicting the outcome of rectal cancer patients undergoing neoadjuvant chemoradiation prior to surgery. 
Methods: Retrospective review of T3/T4, or N+ rectal cancer treated with neoadjuvant chemoradiation 50.4 Gy concurrently with either 5 FU (1 g/m2/d) or Capecitabine 825 mg/m2 twice daily. Four additional cycles of 5-FU chemotherapy (500 mg/m2/d, i.v. bolus) or capecitabine (2500 mg/m2 days 1-14, repeated day 22), were applied post-operatively. Pre-treatment NLR, dNLR, PLR and LMR calculated from peripheral blood cell were compared with clinicopathological parameters. The prognostic value of baseline NLR, dNLR, PLR and LMR for disease free survival (DFS) and overall survival (OS) were assessed using Log rank and Cox regression. 
Results: The final analysis included 80 patients, the receiver operating curve (ROC) calculated cut off values of baseline NLR, dNLR, LMR and PLR in predicting outcome were 3, 2.1, 4.9 and 169 respectively. Elevated NLR, dNLR, PLR, LMR, age of patients (≥50 years), depth of invasion ≥T3, lymph node N1-N2, stage III, grade 3 tumors, and partial response to preoperative chemoradiation were significantly associated with decreased OS, and DFS. Multivariate analysis revealed that elevated NLR and dNLR were independent Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation factors for worse OS and DFS hazard ratio (HR) 2.34 (95% CI=3.41-7.24), 4.53 (95% CI, 2.61-8.32) and DSF with (HR) 1.84 (95% CI=2.27-5.36), 4.23 (95% CI=3.49-9.52) respectively. 
Conclusion: The baseline NLR, dNLR, LMR and PLR showed a significant association with different clinicopathological prognostic factors in rectal cancer patients receiving preoperative chemoradiation. Additionally, NLR, dNLR may be considered as potential independent prognostic indicators of clinical outcomes.


Introduction
The fundamental curative treatment of rectal cancers which comprised one third of the whole colorectal cancer pool, is surgery that entailed total mesorectal excision [1,2]. Neoadjuvant chemo-radiotherapy is the authorized adjunctive modality in mesorectal fascia boundary-menacing rectal cancer. This method considerably ameliorated purging circumambient resection-sidelines consequently substantially minimizing risk of confined relapse [3][4][5][6]. The conventionally practiced extrapolative approaches based on presenting tumor burden were proved to be extremely restrictive in forecasting the outcome due to incongruity of prognosis in patients that were categorized to belong to the same clinical disease stage[7-9]. Additionally, the tremendous inconsistence in response to preoperative chemo-radiation (nCRT) amplified the necessity to explore innovative prognosticators that can accurately anticipate the destiny of rectal cancer patients . Lately, several peripheral blood indicators such as the neutrophil to lymphocyte ratio (NLR), derived neutrophil to lymphocyte ratio (dNLR), platelet to lymphocyte ratio (PLR) and lymphocyte to monocyte (LMR) have been authorized as extrapolative indicators in various kinds of cancers [12,13]. An amplified NLR was linked to abysmal consequences in colorectal cancer [14]. Meanwhile, PLR was perceived to be unreliable as a prognosticator based on its contradictory impact of outcomes as reported by the available body of evidence [15][16][17][18]. Nevertheless, antecedent researches scrutinized on the interaction of a maximum two of inflammatory cells driven prognosticators with outcmes [19][20][21]. Further to this, the optimal The process was initiated after approvals were acquired from the Institutional Board Approval (IRB). The participants agreed to the study through informed consent, which they signed for. The records were reviewed to gather all clinical-pathological date of the rectal cancer patients managed with preoperative chemo-radiation. All laboratory work ups were revised to record the designated scores of pretreatment differential blood cells in addition to platelet counts. The extrapolative indicators neutrophil count to lymphocyte count (NLR), derived neutrophil to lymphocyte ratio (dNLR) was constructed as follows: dNLR= neutrophil count to (white cell count-neutrophil count), platelet to lymphocyte ratio (PLR) and lymphocyte to monocyte ratio (LMR) were calculated.
We selectively included pathologically confirmed clinically staged T3/T4 and /or node positive rectal cancers. All participants the course of preoperative chemo-radiation that entailed 50.4 Gy in1.8 Gy fractions as whole pelvic radiation with concomitant 5-Fluorouracil (1 g/m2/d) as sustained 120 hours infusion during first and fifth weeks of radiation, or 5-Fluorouracil ( 400 mg/m2/d) and leucovorin 20 mg/ m2 intravenous bolus for 4 days during the first and fifth weeks of radiation. Alternatively, Capecitabine 825 mg/m2 twice daily five days per week concurrent with radiation. Total mesorectal excision (TME) surgery was performed 4-6 weeks after conclusion of preoperative CRT. Four additional cycles of 5-FU chemotherapy (500 mg/m2/d, i.v. bolus) or capecitabine (2500 mg/m2 days 1-14, repeated day 22), were applied postoperatively."

Assessment of Response to nCRT
RECIST criteria 1.1. was used to check radiologic response to therapy. It was outlined to be both primary tumor and lymph nodes downstaging as per pre and post neoadjuvant treatment MRI [22]. Total mesorectal excision (TME) was performed in all patients, however extent of surgery whether low anterior or abdominoperineal resection was based on the initial tumor location. Surgery were performed 4-6 weeks after completion of neoadjuvant CRT. Only patients with R0 resection were included. R0 resection was defined as removal of all gross tumor and histopathologic examination of proximal, distal, and circumferential margins that revealed the absence of malignant cells more than 2 mm from the edge. On the contrary, we exempted patients who suffered from disseminated disease and those who had microscopic (R1) or macroscopic(R2) remaining disease postoperatively." Follow up periods including (clinical exam, lab tests, imaging and endoscopy), stood between the 3 to 50 months and were arranged at 4-6 months interval.

Statistical Consideration
To inaugurate the extent of influence among intersected clinicalpathological variables including the verified prognosticators either Mann-Whitney U test (between 2 groups) or Kruskal-Wallis test (≥3 groups) were conducted. The receiver operator curves were devised to uncover the optimal thresholds of studied indicators in anticipating survival times. An area under the curve (AUC) of 1.0 would implied a significant test.
"Statistical informative levels stood at P<0.05. Log-rank test and Cox regression analysis were executed to associate clinical and pathological parameters to treatment outcomes. All analyses were performed using SPSS 16.0 package program, (SPSS, Chicago, IL)."
Different clinical-pathological elements were linked with the tested prognosticators. Correspondingly, the stage III denoting larger tumor sizes and more extensive infiltration of draining nodes were strongly interconnected to augmented scores of NLR, dNLR , LMR and PLR compared to lesser tumor burden tumors (Table 2)." It is worth mentioning, that processing of postoperative specimens confirmed total disappearance of malignant cells from primary tumor bed and regional nodes (path CR) in 12.5% or 10 patients, whereas remaining malignant cells (path PR) were retrieved in (80%) or 64 participants. Therefore, the global response percentage stood at 92.5% i.e. 74 patients, whereas the leftover 6 patients, who accounted for 7.5%, demonstrated progressive or stable disease after preoperative chemoradiation. All details of down staging were displayed in (Table 3). The median number of nodes examined in the 80 TME specimens was 16 (range, 6 to 22 nodes). The median number of nodes with carcinoma was 4, and the median number of cancer-free nodes was 12." Several factors were ensuring pathological complete response as Tstage baseline, N stage baseline, and histopathological grade. The superior response was interconnected with lesser stage (P=0.002) and reduced baseline prognosticators NLR (P= 0.001) , d NLR(P= 0.002), LMR (P=0.001) and PLR(P=0.003), respectively (Table 4).
During follow up period, 32.5% or 26 patients were found to have distant metastasis or local recurrence. From this pool, 27.5% or 22 died from cancer-related complications. The OS and DFS median stood at 28 and 24 months, in that order. Further, the three-year overall survival stood at 72.5% while the disease-free survival registered at 67.5% (Figures 2 and 3). The detailed analysis of survival established that advanced stage, partial response to preoperative chemo-radiation (Figures 4 and 5) and augmented NLR (≥3), ( Figures 6 and 7), dNLR (≥2.1) (Figures 8 and 9) , PLR (≥169), LMR (≥ 4.9) exhibited a strong link to worsened OS, and DFS (

Discussion
The mediators instigated in tumor microevitoment through interaction with host native immune system orchestrates the chains of conditioning and sensitizing interaction of inflammatory cells both in tumor milieu and systemically in peripheral blood that reflect the proliferation of carcinogenesis. For instance, NLR, d-NLR, PLR and LMR that represented systematic indicators of inflammatory reactions induced by colorectal (CRC)malignancies in addition they operated as forecasters of outcome in CRC patients [23,24].Consequently , the current work fundamental target was to emphasize the influence of baseline NLR , dNLR, PLR and LMR prognosticators in conjecturing the prognosis of rectal cancer patients undergoing preoperative chemo-radiation .The baseline NLR , dNLR , LMR and PLR forecasted DFS with informative areas under the curve( AUC) of 0.778 , 0.740, 0.612and 0.545 respectively . Similarly, Ying et al reported that preoperative NLR, d-NLR, PLR are robust prognosticators for colorectal cancer patients with ROC (AUC) of 0.764 , 0.672, 0.727 respectively . However, baseline LMR was exempted due to its nonsignificant AUC of 0.234 [25]. The discordance about the influence of baseline LMR can be principally attributed to the structure of studied population as we encompassed exclusively rectal cancer patients, while Ying et al enrolled both colon and rectal cancer patients in their analysis. Moreover, larger tumor burdens were strongly interconnected to augmented scores of NLR, dNLR, LMR and PLR compared to lesser tumor encumbrance. Ying et reached a resembling conclusion that accentuated baseline prognosticators were strongly attributed to advanced disease [25]." As far as the authors of this study know, this is the first of its kind to successfully isolate cut off values for baseline biomarkers. "More importantly, superior responses were interconnected with lesser stage (P=0.002) and reduced baseline prognosticators NLR (P= 0.001), d NLR (P= 0.002), LMR (P=0.001) and PLR(P=0.003), respectively. It is imperative to mention that the results indicated advanced stage, partial response to preoperative chemo-radiation and augmented NLR (≥3), dNLR (≥2.1), PLR (≥169), LMR (≥ 4.9) exhibited a strong link to worsened OS, and DFS. On the other hand, multiple regression analysis showed that augmented baseline NLR, dNLR along with advanced TNM stage at diagnosis had independent correlation to shortened OS and DFS." In the same vein, Ying et al reported that elevated NLR, poorly differentiated tumors and larger tumor burdens stage were strongly interconnected with compromised OS and DFS [25]. The concordance established between the consequences of the current study and the prosperous body of evidence, emphasized on the forecasting influence of baseline prognosticators NLR, dNLR in rectal cancer patients curatively treated with preoperative chemo-radiation  [ 26,30]. The interaction of neutrophils with distinct malignant cell populations can be provoked by interleukin-6, tumor necrosis factor alpha and granulocyte colony-stimulating factor, myeloid growth factors. It subsequently stimulated an army of cytokines and effector molecules, such as circulating vascular endothelial growth factor (VEGF) which enhanced tumor angiogenesis, growth and metastasis [31][32][33][34][35][36][37]. Moreover, the tumor induced neutrophils amplification can suppress native cellular immunity by suppressing tumoricidal activity of cytotoxic CD8+ Tcells and accentuating T regulatory cells resulting in tumor proliferation [38,39].
The eloquence of the current work resides essentially on emphasizing the role of NLR, dNLR, PLR and LMR as potent prognosticators in rectal cancer managed with preoperative chemo-radiation. Moreover, the forecasting potential of the tested prognosticators was confirmed in a homogeneous population receiving consistent preoperative and surgical procedures. However, the retrospective scheme and limited number of participants were major contenders for absolute validation of our results and they necessitated further verification in a properly designed randomized trial.

Conclusion
The baseline inflammatory forcasters revealed substantial link to various prognostic clinic-pathological parameters in the context of rectal cancer patients who had undergone neoadjuvant chemoradiation. Moreover, both NLR and dNLR can be seen as possible independent predictors for prognosis in the given patient group.7.