COVER LETTER

January 07th, 2019

Dear Editors

I would like to submit the manuscript entitled “Inflammation-Based Scores as a Prognostic

Factor For Survival in Pediatric Osteosarcoma” by dr. Mururul Aisyi, Sp.A(K) to be
considered for publication as a research article in Indonesian Journal of Cancer.

Osteosarcoma is the most common bone malignancy in childhood and adolescence. Systemic
inflammation responses have been associated with cancer development and progression.
Inflammatory-based scores such as : neutrophil-to-lymphocyte ratio (NLR), platelet-to-
lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), and neutrophil-to-platelet
score (NPS) have been proposed to predict clinical outcome in many type of cancer. Despite
substantial improvement which has been documented worldwide, the number of data about
the characteristic and prognosis of osteosarcoma in Dharmais National Cancer Hospital
remain low. Our result showed that there were no association between NLR, PLR, and LMR
with time of event (death). Meanwhile, NPS was significantly associated with failure of
treatment. Furthermore, high NPS was an independent predictor of poor outcome in pediatric
osteosarcoma patients. We believe these findings are relevant and will be of interest to the
readers of your journal.

We declare that this manuscript is original, has not been published before and is not currently
being considered for publication elsewhere.

We wish to confirm that there are no known conflicts of interest associated with this
publication, and there has been no significant financial support for this work that could have
influenced its outcome. As Corresponding Author, I confirm that the manuscript has been
read and approved for submission by all the named authors.

We hope you find our manuscript suitable for publication and look forward to hearing from
you in due course.

Sincerely,

dr. Mururul Aisyi, Sp.A(K)

Pediatric Hematology Oncology Department, Indonesian NCC, Dharmais Cancer Hospital
11420
Tel/Fax: 085757149704
Email: aci97nabil@yahoo.com

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Title :

Inflammation-Based Scores As a Prognostic Factor for Survival in Pediatric

Osteosarcoma

Authors:

Mururul Aisyi2 Nur Asih1, Rizky Hutomo2 Agus Kosasih3 Achmad Basuki4 Ayu
Hutami Syarif2 Aditya Bayu1

Affiliations:

1) Pediatric Hematology Oncology Department, Indonesia National Cancer Center

Dharmais Cancer Hospital, Jakarta, Indonesia
2) General Practitioner, Indonesia National Cancer Center Dharmais Cancer Hospital,
Jakarta, Indonesia
3) Clinical Pathology Department, Indonesia National Cancer Center Dharmais Cancer
Hospital, Jakarta, Indonesia
4) Orthopedic Surgery Department, Indonesia National Cancer Center Dharmais Cancer
Hospital, Jakarta, Indonesia

Corresponding Author:

Mururul Aisyi
Address: Pediatric Hematology Oncology Department, Indonesia National Cancer
Center Dharmais Cancer Hospital, Jakarta, Indonesia
Email: aci97nabil@yahoo.com

Abstract

Background:

Osteosarcoma is the most common bone malignancy in childhood and adolescence. Systemic
inflammation responses have been associated with cancer development and progression.
Inflammatory-based scores such as : neutrophil-to-lymphocyte ratio (NLR), platelet-to-
lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), and neutrophil-to-platelet
score (NPS) have been proposed to predict clinical outcome in many type of cancer. Despite
substantial improvement which has been documented worldwide, the number of data about
the characteristic and prognosis of osteosarcoma in Dharmais National Cancer Hospital
remain low. The aim of this study is to describe the characteristics and evaluate prognosis of

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pediatric osteosarcoma in Dharmais National Cancer Hospital based on the value of NLR,
PLR, LMR, and NPS. This study collected data from medical records of 41 patients
hospitalized during the period 2010-2017. Patients were selected using inclusion and
exclusion criteria. NLR, PLR, LMR, and NPS were measured from peripheral blood counts
and compared each other. Survival (OS) was plotted using Kaplan-Meier Method and
evaluated by the log-rank test. Our result showed that there were no association between
NLR, PLR, and LMR with time of event (death). Meanwhile, NPS was significantly
associated with failure of treatment (p<0.05). Furthermore, high NPS was an independent
predictor of poor outcome compared to both low NPS (HR=3.94,95%CI = 0.94-12.76,
p=0.04) and medium NPS (HR=6.26,95%CI=1.30-26.40,p=0.012).

Introduction :

Osteosarcoma is the most common malignancy of bone which primarily afected primitive
bone-forming mesenchymal cells. Osteosarcoma was agely distributed in bimodal pattern,
reaching peaks in adolescence (10-14 years) and older adulthood ( older than 65 years). The
5-year survival rate for children dramatically increased after introduction of adjuvant
chemotherapy with localized osteosarcoma from 10% to about 60%-80% and for metastatic
osteosarcoma to 15%-30%. Many studies reported that ciculating Serum Amyloid A,
Alkaline Phospatase, T cell immunoglobulin and mucin domain-3 (Tim 3), and CXC
Chemokine Ligand 4 (CXCL4) can be used as predictors of osteosarcoma prognosis.
However, these biomarkers are still unaffordable for developing country like Indonesia due to
their high cost. Besides, the vulnerability of the research method made them
underinvestigated that require further exploration.

Inflammation-based score from complete blood count has been exploited to predict the
prognosis in many cancer, including osteosarcoma. It was known that the increase of
neutrophils and decrease of lymphocyte count as a result of systemic inflammatory response.
Inflammatory microenvironment promotes angiogenesis and metastasis, subverts adaptive
immune responses, and alters responses to hormones and chemotherapeutic agents. Due to
the association of inflammation in cancer development, the prognostic significance of several
inflammation biomarkers and hematological indices, including neutrophil-lymphocyte ratio
(NLR), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR), and neutrophil-
platelet score (NPS) have been investigated in osteosarcoma. In this study, we aimed to
evaluate survival rate of pediatric osteosarcoma based on inflammatory markers in Dharmais
national Cancer Center.

Method:

Patients

This retrospective cohort study involved 41 children with osteosarcoma during 2010-2017.
Ethical clearance was approved by institutional review board. Demographic and
clinicopathologic data (age, gender, disease classification, tumor site, metastasis,
chemotherapy, complete blood count) were obtained from medical records while time to

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death data were collected by interviewing relatives by phone in january-april 2018. Follow up
time varied from 3 months to 72 months.

Variable Definition

Disease stadium was grouped as II B and III based on Enneking Staging System. Metastatic
disease were also taken into account. Additionally, the location of tumor was separated into
upper long bones and lower long bones. To assess survival outcome, dead patients were
categorized as failed. Neutrophil, lymphocyte, platelet, monocyte count were obtained and
calculated to measure the NLR, PLR, LMR, and NPS. The NLR was derived by dividing the
neutrophil count by the lymphocyte count and separated into high NLR (HNLR) and low
NLR (LNLR) based on cut off value; the PLR was derived by dividing the platelet count by
the lymphocyte count and grouped as high PLR (HPLR) and low PLR (LPLR); the LMR
was obtained by dividing the lymphocyte count by the monocyte count and categorized into
high LMR (HLMR) and low LMR (LLMR); and the NPS was resulted from combining the
absolute neutrophil count and platelet count and divided into high NPS, medium NPS, and
low NPS. The cut off value for them were 2, 150, 3.43, ANC 7.5 and thrombocyte >400,
respectively.

Statistical Analysis

Data were analyzed statstically with Statistic Package for Social Service (SPSS) 20.00 and
STATA software. All those parameters were compared each other in univariate analysis.
These categorical variables were expressed as median, mean, and percentages. Bivariate
analysis determined the correlation between NLR, PLR, LMR, NPS and status of event which
expressed in p value. Only significant variables (p<0.05) were inserted into hazard model.
Predictors for survival outcome were tested with multivariate log regression test and results
were described as hazard ratio (HR) and 95%CI. Overall survival was analyzed by Kaplan-
Meier method by inserting time of death, status of event, and independent variables and
evaluated with log-rank test.

Result:

Patients Characteristics

A total of 41 children enrolled in this study consisted of 20 boys and 21 girls with median
age of 12 years and ranged from 8 to 18 years. About 56,1% patients were in stadium II B at
the time of diagnosis and over four-third of the patients (80.5%) were reported to be dead at
follow up. Furthermore, it is clearly described in the table 1 that lower long bones were
predominant site for osteosarcoma (68.3%) and half of the patients showed no metastasis
(56.1%).

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Table 1. Patient caharacteristics and associations with time of event (death). Data presented
are number (%) and expressed in p value

Variables Frequency Time of Event P value

Yes No
Gender 0.408
Female 21 (51.2%) 17 4
Male 20 (48.8%) 16 4
Age
<10 4 (9.8%) 4 0 0.585
≥10 37 (90.2%) 29 8
Tumor Site
Upper long bones 9 (22%) 7 2 0.048
Lower long bones 28 (68.3%) 22 6
Others 4 (9.8%) 4 0
Stadium 0.245
II B 23 (56.1%) 18 5
III 18 (43.9%) 15 3
Metastasis 0.245
Yes 18 (43.9%) 15 3
No 23 (56.1%) 18 5
NLR
HNLR 17 (41.5%) 12 5 0.755
LNLR 12 (29.3%) 11 1
PLR
HPLR 14 (34.1%) 11 3 0.400
LPLR 15 (36.6%) 12 3
LMR
HLMR 21 (51.2%) 18 3 0.924
LLMR 8 (19.5%) 5 3
NPS
HNPS 4 (9.8%) 4 0 0.028
MNPS 11 (26.8%) 6 5
LNPS 14 (34.1%) 13 1
HNLR : high neutrophil lymphocyte ratio, LNLR : low neutrophil lymphocyte ratio, HPLR : high platelet
lymphocyte ratio, LPLR : low platelet lymphocyte ratio, HLMR : high lymphocyte monocyte ratio, LLMR : low
lymphocyte monocyte ratio, HNPS : high neutrophil lymphocyte ratio, LNPS : high neutrophil lymphocte ratio

Table 1 showed that tumor location was statistically associated with failure. Similarly, NPS
value was observed to have a clear correlation with failure as well. In contrast, other variables
had no correlations to failure in osteosarcoma.

Survival Analysis

The median overall survival of all patients was 10 months (range 2.1 to 17.8 months )
months. Eight patients (19.5%) patients were alive at the end of the follow up period. The
survival curve revealed that the median overall survival of patients with tumor in upper long
bones was 24 months whereas in lower long bones was 5 months. Furthermore, high NPS had
significant shorter overall survival than patients with medium and low NPS (mean OS, 4.6 vs

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24.6 months, 4.6 vs 17.9 months, respectively p=0.028). Therefore, High NPS was an
independent predictor of poor outcome compared to both low NPS (HR=3.94, 95% CI=0.94-
12.76, P=0.04) and medium NPS (HR=6.26, 95% CI=1.30-26.40, P=0.012)

(A) (B)

(C)
Figure 1. Kaplan Meier survival curves for overall survival according to tumor site and NPS
in 41 patients. (A) Overall Survival children with osteosarcoma; (B) Overall Survival based
on tumor site; (C) Overall Survival based on NPS score

Discussion

The accuracy of prognostic evaluation is essential for treating osteosarcoma. Current

predictive factors, including metastasis, tumor site, age, and metastasis were established in
many studies. Additionally, systemic inflammation markers have been explored and can be
used to determine prognosis and survival of osteosarcoma patients. As the peripheral blood
count test is routinely performed without additional effort, it is simple, reproducible, and
inexpensive to measure the response of inflammation and prognosis.

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Huang et al reported in retrospective study involved 162 cases that neutrophil count was an
independent prognostic factor among other inflammatory parameters. This result was
confirmed by Vasquez et al reported that pre-treatment NLR was associated with overall
survival in pediatric sarcoma, including osteosarcoma. However, our study found there was
no association between NLR ratio with time of event and patients survival.

Post-operation PLR ratio which was evaluated by et al showed no corelation with overall
survival and thus unable to predict the prognosis. This prior reult has been verified by our
study. Meanwhile, low LMR was associated with poor outcome in osteosarcoma but failed to
be risk factor for death in multivariate analysis.

There were many studies reported about NPS in differet type of cancer such as colorectal
cancer, head and neck, and renal cancer but no study have investigated in osteosarcoma.
Although it was not in the same context, current study supported previous result that NPS
significantly associated with poorer cancer survival.

It has been proposed by virchow that chronic inflammation contributes anda predsiposes
tissue to cancer development. Studies have shown that proinflammatory cytokines and
chemokines which regulate the recuruitment of leukocytes are frequently found in tumor
microenvironment. Elevated counts of myeloid cells such as neutrophils reflect a
proinflammatory response and thus stimulate angiogenesis whereas the adaptive immune
system is converted. The inflammatory response result in neutrophilia, leukocytosis,
thrombocytosis, and lymphocitopenia. The combination of high neutrophil count and platelet
count suggested that there is an increase in inflammatory response contributing to poorer
survival rate in osteosarcoma. This response represents progressive celluar breakdown and
remodellig of tissue (proliferation of tumor cells). It is known that enzymes within
neutrophils such as myeloperoxidase, interleukin-6 (IL-6), defensins, lysozyme and
collagenase and platelet deived factors which is vascular endotelial growth factor (VEGF)
act to induce angiogenesis and to help tumor escape from immune system.

The present study has some limitations. Firstly, there is no exact measurement time for
complete blood count. Different obtaining time might affect result in the value of
inflammatory markers. Secondly, data from other factors such as concurrent drugs
consumption and comorbidities may confound nutrophil of platelet levels and were not
available in this study.

CONCLUSION

Our results showed that there were no associations between NLR, PLR, LMR and time of
event (death). Compared to other prognostic factors, NPS can predict survival in
osteosarcoma patients. Spesifically, high NPS (HNPS) was an independent predictor of poor
outcome in pediatric osteosarcoma.

ACKNOWLEDGMENT

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The authors gratefully acknowledge the support of Medical Record Unit of Dharmais
National Cancer Center, particularly......

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