Usefulness of neutrophil-lymphocyte ratio and platelet-lymphocyte ratio as a predictor of disease-free survival in breast cancer : A cross-sectional study

The relationship between neutrophil-lymphocyte ratio (NLR) Background: with outcome is a complex issue. A high NLR reflects systemic inflammation. This study aimed to estimate the relationship between NLR, and platelet-lymphocyte ratio (PLR) in disease-free survival (DFS). This was a cross-sectional study in which we reviewed the Methods: patient files of 102 patients with breast cancer treated at the Babylon Oncology Center from January 2009 to September 2014, who had follow-up for at least 36 months. The following data were collected from patient files: age, diagnosis date, date of recurrence and/or metastasis, follow-up, histological tumor type, tumor size, node metastasis stage, histological differentiation degree, estrogen and/or progesterone receptor expression, HER2 neu status, and metastasis site. The mean age of patients was 50.4 ± 11.7 years and lowest Results: period of follow up was 40 months. Longest DFS was 62 months, with 5 years DFS in 52.5% of patients. Stage N0 was associated with a significantly higher DFS compared to stage N1. Isolated local recurrence was seen in 15% of patients and combined local recurrences with distant metastasis was observed 37%. NLR had the highest discrimination ability to predict recurrence and distant metastasis. An increase in NLR was associated with poor DFS, and it can Conclusion: therefore be a predictive and prognostic factor. NLR’s established prediction model warrants further investigation.


Reviewer Status
Invited Reviewers version 1

Introduction
Worldwide, breast cancer is the most diagnosed cancer type among women 1 . Animal models suggest "immune-editing" in which activation of immune mechanisms control the tumor, but over time lead to the selection of tumor cells that escape the immune pressure and grow progressively 2 . Most tumor antigens identify as non-mutated self-antigens. Tumors are heterogeneous, and the antigens on cells of one tumor are variable, even within the same patient, so the down-regulation of major histocompatibility complex molecules and other components of the antigen-presentation process can occur 1 . Tumors also do not express the ligands recognized by innate immune cells that microbes express or the co-stimulatory ligands necessary to stimulate adaptive T cells 2 . The expression of Fas ligand by some tumor cells help to maintain a state of immune privilege that induce apoptosis. Tumor cells lead to the release of many cytokines and soluble factors, such as prostaglandin E2, that are not conducive to antitumor immunity. Cancer-associated factors have been shown to inhibit the production and stimulatory capacity of tumor cells 3 . T-helper cell responses skewed toward a Th2 phenotype, lead to inhibition of the Th1 response and cellular immunity that mediates tumor rejection 4 .
The evidence of the relationship between inflammation and cancers prognosis has increased in past years, especially gastrointestinal tumors and non-small-cell lung cancers, and even breast cancers. The systemic inflammatory responses may mimic biochemical or hematological markers, such as raised C-reactive protein and the elevation of white blood cells, neutrophils, and platelets. Elevation of neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) in breast cancer patients is not well studied in Iraq. This relationship is a complex and multifactorial process that is still poorly understood, but a high NLR may reflect systemic inflammation in enhancing angiogenesis, tumor growth and development of metastasis. Therefore, this study aimed to estimate the relationship between NLR, PLR and disease-free survival (DFS) in breast cancer patients in Iraq.

Study design and setting
This is a cross-sectional retrospective study. Breast cancer patients who were treated at Babylon Oncology Center, Baghdad, Iraq from January 2009 to September 2014 were included in this study. The data was collected between May 2017 and April 2018.
Exclusion criteria: Locally advanced and metastatic breast cancer (T3-T4, N1-N3, M1); neoadjuvant treated patients; patients with data lacking for follow-up and pretreatment CBC with differential count; known causes of neutrophilia (those who already have this disease such as infections, inflammation, burns, heart attack, and drugs, e.g. steroids).

Data sources
The following data were collected from patient files: age, diagnosis date, date of recurrence and/or metastasis, follow-up, histological tumor type, tumor size, node metastasis stage, histological differentiation degree, estrogen and/or progesterone receptor expression, HER2 neu status, and metastasis site.

Statistical methods
The statistical analysis used the following tests: Anderson darling test, a statistical test of whether a given sample of data is drawn from a given probability distribution; Kaplan-Meier analysis, a non-parametric statistic used to estimate the survival function from lifetime data, which was used to assess DFS; hazard ratio (HR), the ratio of the hazard rates corresponding to the conditions described by two levels of an explanatory variable; ROC curve, a performance measurement for classification of problems at various thresholds settings; sensitivity analysis for test quality; and specificity analysis for test extension. SPSS 20.0 software package was used to for statistical analysis. P-value of <0.05 was considered significant. Patients who had missing data for variables were excluded from the analysis.

Ethical considerations
The

Results
Out of a total of 1167 case files only 102 patients fit the eligibility criteria and completed the study, with a mean age of 50.4 ± 11.7 years (range, 23-75 years, the rest of the disease characteristic illustrated in (Table 1). Local recurrence had the lowest rate, while combined local and distant recurrence had the highest rate ( Table 1). The median DFS was 62 months with 5 years DFS in 52.5% patients, patients with stage N0 had a significantly higher DFS compared to stage N1, and those patients with a positive hormonal status have a significantly better DFS compared to those with a negative hormonal status (Table 2; Figures 1A-D). NLR had the highest discrimination ability to predict recurrence (since AUC between 0.7 -0.79), while the rest of the variables show poor discrimination ability, as illustrated in Table 3 and Table 4 and Figures 1E and F. NLR fair specificity (76.9%) with lower sensitivity (62.2%), with optimal cut point of >2.194 to predict all recurrence. NLR also showed similar predictability for distant metastasis, while for local recurrence NLR had poor ability to predict local recurrence (Table 5 and  Table 6; Figure 1G and Figure 2). Table 7 shows uni-and multivariate analysis of predictors of DFS with a significant p-value (p=0.007) for NLR, which means that it is an independent risk factor ( Figure 3).

Discussion
This is the first study to show an association between high NLR and poor prognosis in Iraqi breast cancer patients. A higher NLR independently reflected a higher risk of local recurrence       and distant metastasis in women with early breast cancer with a cutoff point of 2.194.
We found significant differences in DFS (16 months) according to our NLR cut off point with significant p-value (p=0.004). The role of lymphocytes in cancer is exemplified by the strong association between high densities of tumor-infiltrating lymphocytes and better responses to both cytotoxic treatments and outcome in patients 5,6 . Two meta-analysis studies have confirmed the association between elevated NLR and poor prognosis for breast cancer 7,8 ; however, studies about these values are rare and not done in Iraq. In this study, we validated the usefulness of high NLR in early stage breast cancer up to stage IIA and to estimate DFS for at least 36 months follow-up.
In our univariate and multivariate analysis, we found hazard ratio (HR) for NLR 2.5 with significant p-value (p=0.007), while in the univariate analysis the nodal status and hormonal status were significant as dependent prognostic factors. Comparing our result with Ethier et al., a meta-analysis which comprised patients with reported HRs for DFS, and included only non-metastatic cases 8 , our result has the same significance with good sample size and follow-up period; however, we couldn't calculate overall survival (OS). Although another study shown that PLR was not related with DFS or OS in women 9 , in our study PLR was neither sensitive nor specific with non-significant p-value, so it will not considered as a prognostic index.

Conclusion
The role of NLR is a prognostic marker and elevated NLR is correlated with poor DFS in early breast cancer patients.
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Grant information
The author(s) declared that no grants were involved in supporting this work. 1.

2.
Are the conclusions drawn adequately supported by the results?

Partly
No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
In this study, Al-Bairmany attempt to explore the effect of neutrophil-lymphocyte ratio (NLR) and et al platelet-lymphocyte ratio (PLR) on Disease Free Survival (DFS) in patients with breast cancer. In this retrospective study, the authors find that high NLR correlates with poor DFS, suggesting its use as a prognostic marker in breast cancer. While the study attempts to address an important question, that of a clinically unmet challenge of finding robust biomarkers that can predict/prognosticate breast cancer survival, the manuscript is poorly written and referenced, the results are inadequately presented with not enough information in either the methods, figure legends or the plots to derive and interpret the information and results. While the authors claim that the prognostic role of NLR is controversial, they do not discuss the controversy adequately either in the introduction or discussion. For specific comments, please refer to the point-by-point review below.
Many Grammatical errors, choice of words and awkward sentence structure make it very difficult to read through and review and manuscript, starting from the abstract. We advise the authors to kindly revise the manuscript thoroughly and correct the language such that the reviewers can focus on critiquing the data rather than the linguistic shortcomings of the manuscript. Additionally, several references are missing (as highlighted in the comments below) making It difficult to get a solid understanding of the background through cross-referencing. Also, a lot of these references are outdated and the field has progressed profoundly in the last few years. The authors need to thoroughly review recent literature.

Abstract:
Please amend sentence to: "the relationship between neutrophil-lymphocyte ration (NLR) and outcome is controversial". Also, do the authors mean Survival outcome? If yes, please clarify.
Please amend to: "this study aims to explore the relationship between NLR…". Also, the authors 3.
8. Figure 1B: Is this correct that patients with stage T1 tumors with less aggressive tumors have worse prognosis than stage 2, which is more aggressive? Please explain this outcome. Without knowing what these are how can we interpret the robustness of the data or appreciate the differences in survival? Figure 1G: Why is the red curve cut short? One would expect a tail with patients that have an NLR of greater than 2.194?
Also, how was the cutoff value of 2.194 determined? This information is missing from Methods.
Neutrophil-lymphocyte ratio or NLR: were these derived from blood samples or biopsies? As demonstrated in other cancers such as brain tumors,the presence of neutrophils in the blood versus in the tumor can predict response to treatment. The authors need to mention in their methods where the neutrophils, lymphocytes and platelets are derived from (blood, biopsy etc) and explain how they were quantified.
Also, the patients who were included in the study, are they treated in any way i.e. surgery, radiation and chemotherapy? The therapeutic strategy also affects DFS and as such should be explicitly stated by the authors.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: Cancer Biology, treatment resistance, tumor recurrence, cell biology, molecular biology, biochemistry, breast cancer, brain cancer, mechanobiology, tissue tension We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above.