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\begin{document}

\title{Biological risk based on preoperative serum CA19-9 and histology grade
predicts prognosis in patients with pancreatic cancer}



\author[1]{Shaofei Chang}%
\author[2]{Yaohua Liu}%
\author[1]{Yuexiang Liang}%
\author[1]{Quan Man}%
\author[1]{Haorui Li}%
\author[1]{Yu Guo}%
\author[1]{Tiansuo Zhao}%
\affil[1]{Tianjin Medical University}%
\affil[2]{Shanxi Medical University}%


\vspace{-1em}



  
  \date{June 01, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background:} Preoperative serum CA19-9 and histology grade could
reflect the biological characteristics of pancreatic ductal
adenocarcinoma (PDAC). This study aims to explore the combined effect of
preoperative CA19-9 and histology grade on the prognosis of patients
with PDAC. \textbf{Methods:} A total of 612 patients with PDAC
undergoing curative pancreatectomy were retrospectively enrolled. A
biological risk model was established based on preoperative CA19-9 and
histology grade. Prognostic significance of the biological risk was
evaluated. \textbf{Results:} 360 (58.8\%) patients had preoperative
CA19-9\textgreater{}112 U/ml and 348 (56.9\%) patients had high
histology grade. Biological risk based on preoperative CA19-9 and
histology grade was independently associated with survival of PDAC
patients. The biological risk was incorporated into the eighth edition
of the TNM staging system and a modified TNM (mTNM) staging system was
developed. The ROC curves showed that the area under curve(AUC) of the
mTNM staging system was significantly greater than that of the TNM
staging system. \textbf{Conclusion}: Biological risk based on
preoperative CA19-9 and histology grade was an independent prognostic
factors for patients with PDAC. Incorporating the biological risk into
the TNM staging system could improve the the accuracy of the TNM staging
system in predicting prognosis of PDAC.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal
malignancies. According to the World Health Organization, the estimated
number of new cases of pancreatic cancer ranked 12th among all malignant
tumors in 2020\textsuperscript{{[}1{]}}. The 5-year survival rate was
approximately 11.5\%\textsuperscript{{[}2{]}}. Multiple disciplinary
therapy based on the tumor stage and biological characteristics of
primary lesion is highlighted in improving the survival of patients with
PDAC. Therefore, it is vital to classify PDAC more accurately.

The American Joint Committee on Cancer (AJCC) tumor-node-metastasis
(TNM) staging system is the most commonly used classification for the
prognostic evaluation and treatment decision of
PDAC\textsuperscript{{[}3{]}}. The TNM classification~has undergone
continuous improvement to reflect the current understanding of the
extent of disease\textsuperscript{{[}4{]}}.~Several studies have
clarified the accuracy of the TNM staging of Pancreatic Cancer (8th ed.,
2017) \textsuperscript{{[}5,6,7{]}}, in contrast, several other studies
demonstrated no survival differences between stages of TNM
classification and proposed modified TNM or newly staging
system\textsuperscript{{[}8,9{]}}.

Tumor biology shows significant impact on the prognosis of patients with
malignancy and has been incorporated into the staging system to improve
the accuracy of prognostic prediction\textsuperscript{{[}10,11{]}}.
Since both tumor burden and malignant degree could reflect the
characteristics of tumor biology\textsuperscript{{[}12,13{]}}, we
supposed that combination of the factors which represent tumor burden
and malignant degree would provide a full understanding of tumor biology
on prognosis of cancer, and incorporating factors associated with tumor
biology into staging system could improve the accuracy of the TNM
staging system in predicting prognosis. Carbohydrate antigen 19-9
(CA19-9) is associated with the tumor burden
\textsuperscript{{[}14{]}}and histology grade is associated with the
degree of malignancy\textsuperscript{{[}15,16{]}.} Therefore, the
combination of preoperative serum CA19-9 and histology grade could
reflect the tumor biology of PDAC. In the present study, we
retrospectively analysed the data of 612 patients with PDAC who
underwent curative resections. The aim of this study was to explore the
combined effect of preoperative serum CA19-9 and histology grade on the
prognosis of patients with PDAC.

\textbf{Materials and methods}

This study was approved by the Ethical Review Committees of Tianjin
Medical University Cancer Institute and Hospital, and conducted in
accordance with the ethical guidelines of the Declaration of Helsinki.
Informed consent was waived due to retrospective nature of the study.

\textbf{Patients}

A total of 692 patients with PDAC who underwent curative resection in
the Department of Pancreatic Cancer, Tianjin Medical University Cancer
Institute and Hospital between January 2011 and December 2018 were
eligible for this study. Eligibility criteria included: (I) patients
with PDAC; (II) patients with R0 resection; (III) patients who were
completely followed up. Exclusion criteria included: (I) patients with
history of pancreatectomy or other malignancy; (II) patients with
arterial tumor contact (celiac axis, superior mesenteric artery, or
common hepatic artery); (III) patients with distant metastasis; (V)
patients who accepted neoadjuvant chemotherapy; (VI) patients who died
during the initial hospital stay or 1 month after surgery. After
excluding 10 patients with pancreatic mucinous carcinoma, 9 patients
with pancreatic adenosquamous carcinoma, 1 patient with lung cancer, 1
patient with gastric cancer, 3 patients with breast cancer, 10 patients
with R1 resection, 1 patient with R2 resection, 14 patients confirmed
with distant metastasis during laparotomy, 22 patients lost, 4 patients
accepted neoadjuvant chemotherapy, 2 patients died during the initial
hospital stay and 3 patients died within 1 month after surgery,
ultimately, 612 patients were included in this study.

\textbf{Evaluation of clinicopathological variables and survival}

Clinicopathological variables studied included 14 factors: sex, age at
surgery, preoperative serum CA19-9, preoperative serum carcinoembryonic
antigen(CEA), preoperative serum CA242, tumor location, type of surgery,
TNM stage,T stage, N stage, histology grade, lymphovascular invasion,
perineural invasion and postoperative adjuvant chemotherapy.

The levels of preoperative serum tumor markers (CA19-9, CEA and CA242)
were detected within 1 week before surgery. The normal upper limits of
serum tumor markers were adopted as follows: CA19-9 (37.0 U/ml), CEA
(5.0 ng/ml) and CA242 (20 U/ml). In this study, all patients with total
bilirubin [?] 250mmol/L were treated with percutaneous transhepatic
biliary drainage or endoscopic retrograde biliary drainage. After
biliary darinage, the level of preoperative serum CA19-9 was detected
again.

The pathological diagnosis was established by two professional
pathologists. Tumors were staged according to the
8\textsuperscript{th}edition of AJCC TNM staging of pancreatic cancer.
The histology grade were classified into two groups based on the degree
of tumor differentiation: low grade, including well or moderately
differentiated adenocarcinoma; and high grade, including poorly
differentiated or undifferentiated adenocarcinoma. Lymphovascular
infiltration (LVI) was referred to as blood vessel and lymphatic
invasion. Perineural invasion is an infiltrative process of peripheral
nerves by the primary neoplasm within the immediate vicinity.~Patients
who received more than three cycles of postoperative adjuvant
chemotherapy were defined as chemotherapy group.

\textbf{Follow-up}

Patients were reviewed monthly for 6 months after surgery. Then they
were followed up every 3 moths for up to 2 years after surgery, then
every 6 moths for up to 5 years, and then every year or until death. A
physical examination, laboratory tests including serum CA19-9 and CEA
levels, and abdominal ultrasound were performed at each visit, whereas
chest and abdominal computed tomography or abdominal magnetic resonance
imaging were routinely obtained every 6 moths. For patients with
suspected recurrence or metastasis, imaging examinations were performed
at any time. The OS was calculated from the day of surgery until time of
death or final follow-up. The RFS was defined as the interval from the
surgical time until tumor recurrence or the last follow-up. The median
follow-up were 51 months (range: 1-126) for OS and 49 months (range:
1-126) for RFS, respectively. The date of the final follow-up was March
30, 2022.

\textbf{Statistical analysis}

The receiver operating characteristic (ROC) curves were used to identify
the potential cut-off values of preoperative serum CA19-9 in predicting
survival. The ROC curves were also used to compare the area under curves
(AUC) of the modified TNM(mTNM) staging system and the eighth edition of
the TNM staging system to show the advantage of each in predicting
survival. The OS curves were calculated using the Kaplan-Meier method
based on the length of time between the primary surgical treatment and
final follow up or death.The log-rank test was used to assess
significant differences between curves. Independent prognostic factors
were identified by the COX proportional hazard regression model which
were used to measure homogeneity and discriminatory ability.
P\textless{}0.050 (bilateral) was considered statistically significant.
The statistical analysis was performed using the statistical analysis
program package IBM SPSS 23.0 and MedCalc v.19.6.1.

\textbf{Results}

\textbf{Clinicopathological characteristics of the whole cohort}

The baseline characteristics of the whole cohort is listed
in\textbf{Table I} . The 612 patients included 352 males (57.5) and 260
females (42.5). The age ranged from 31 to 82 years, and the media age
was 61 years. The median levels of preoperative CA19-9, CEA and CA242
were 156.25 U/ml, 3.3550 ng/ml and 19.64 U/ml, respectively. There were
416 patients with PDAC located at the head of pancreas and 196 patients
at body and tail. A total of 414 patients accepted
pancreaticoduodenectomy, 195 patients accepted distal pancreatectomy and
3 patients accepted total pancreatectomy. Of the 264 patients with low
histology grade, 23 patients were well differentiated and 241 patients
were moderate differentiated. The remaining 348 patients who were poorly
differentiated were classified into high histology grade. The median OS
of the entire cohort was 18 months (6-126 months), with survival rates
of 71.3, 43.3, 31.3 and 19.1\% after 1, 2, 3 and 5 years of follow-up,
respectively.

\textbf{Survival analysis of patients with PDAC}

The ROC curves showed that preoperative serum CA19-9 could predict the
prognosis of patients with PDAC (AUC=0.576, 95\%CI:0.535-0.615,
P=0.003)(\textbf{Figure 1} ). The optimal cutoff point of preoperative
serum CA19-9 to predict survival was 112 U/ml. Then, the patients were
categorized into two groups according to the optimal cutoff value of
preoperative serum CA19-9: group 1, in which the level of peroperative
serum CA19-9 was [?]112 U/ml; and group 2, in which the level of
preoperative serum CA19-9 was \textgreater{}112 U/ml.

The results of the univariate and multivariate survival analyses were
presented in \textbf{Table II} . In the univariate analysis, the
following 8 factors evaluated had a significant effect on survival: age
at surgery (\textless{}70 years vs [?]70 years), preoperative CA19-9 ([?]112
U/ml vs \textgreater{}112 U/ml), preoperative serum CEA ([?]5 ng/ml vs
\textgreater{}5 ng/ml), postoperative adjuvant chemotherapy, TNM stage,
histology grade (low grade vs high grade), perineural invasion and
lymphovascular invasion.

PDAC patients with CA19-9 \textgreater{}112U/ml had a significantly less
5-year OS than those with CA19-9 [?]112U/ml (5-year OS: 15.9\% vs 22.6\%,
P\textless{}0.001, \textbf{Figure 2, A} ), and patients with high grade
also had a significant less 5-year OS than those with low grade (5-year
OS: 15.7\% vs 23.5\%, P\textless{}0.001, \textbf{Figure 2, B} ).

In the multivariate analysis, preoperative serum CA19-9 (HR was 1.355
for CA19-9\textgreater{}112 U/ml, P=0.005) and histology grade (HR was
1.545 for high grade, P\textless{}0.001) were found to be independent
prognostic factors for OS, as were age at surgery ([?]70 years), TNM stage
and postoperative adjuvant chemotherapy.

\textbf{Development of biological risk model}

Initially, all patients were categorized into four groups according to
the levels of preoperative serum CA19-9 and histology grade: group 1,
CA19-9[?]112 U/ml and low grade; group 2, CA19-9[?]112 U/ml and high grade;
group 3, CA19-9\textgreater{}112 U/ml and low grade; group 4,
CA19-9\textgreater{}112 U/ml and high grade. The 5-year OS rates were
28.5, 23.0, 18.6 and 11.9\% in group 1, 2, 3 and 4, respectively. There
was no survival difference between group 2 and group 3 (5-year OS:
23.0\% vs 18.6\%, P=0.805) (\textbf{Figure 3, A} ).

Peroperative serum CA19-9 and histology grade were used to build a
biological risk (BR) score taking into account of their hazard ratios in
the multivariate analysis. Patients with preoperative serum CA19-9 [?]112
U/ml and low grade were given a BR score of 0. Patients with
preoperative CA19-9\textgreater{}112U/ml and low grade as well as those
with preoperative serum CA19-9 [?]112 U/ml and high grade were given a BR
score of 1. Patients with preoperative CA19-9\textgreater{}112U/ml and
high grade were given a BR score of 2. The BR score was 0 for 129
patients, 1 for 267 patients and 2 for 216 patients, respectively.
Patients were divided into three groups according to the BR score: low
risk group (BR=0), middle risk group (BR=1) and high risk group (BR=2).
The 5-year OS rates were 28.5\%, 14.9\% and 7.7\% for patients in the
low-risk group, middle-risk group and high-risk group, respectively
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=92.762, P\textless{}0.001)(\textbf{Figure 3, B} ).
In the multivariate analysis, the BR score was an independent prognosis
factor for OS (HR was 1.342, P=0.047). Preoperative serum CA19-9 and
histology grade were not significant if the BR score was included in the
multivariate analysis (\textbf{Table III} ).

\textbf{Incorporation of biological risk into the eighth edition of AJCC
TNM staging system}

With the TNM-stratified analysis, there was no significant survival
difference between low-risk group and middle-risk group in stage I,
while the OS of the two groups was significantly better than that of
high-risk group in stage I (\textbf{Figure 4,A} ). Of the patients in
stage II, the survival differences were significant between each two
groups (\textbf{Figure 4,B} ). Of the pateints in stage III, there was
no significant survival difference among the three groups
(\textbf{Figure 4,C} ). The OS of patients in low-risk and middle-risk
group, who were at stage I was similar to that of patients in low-risk
group who were at stage II. The OS of patients in high-risk group who
were at stage I was similar to that of patients in middle-risk group who
were at stage II. The OS of patients in high-risk group who were at
stage II was similar to that of patients in stage III(\textbf{Figure
4,D} ).

According to the results of the strata analysis, we incorporated the
biological risk into the eighth edition of the TNM staging system and
introduced our modified TNM (mTNM) staging system (\textbf{TableIV} ).
In the mTNM staging system, the 5-year OS rates of stage mI, mII and
mIII were 31.5\%, 17.2\% and 7.2\%, respectively
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=78.603, P \textless{}0.001). In the TNM staging
system, the 5-year OS rates of stage I, II and III were 28.2\%, 14.9\%
and 7.7\%, respectively (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=37.794, P
\textless{}0.001) (\textbf{Table V, Figure 5 A,B} ). Then, the mTNM
stage and the factors associated with OS in the univariate analysis were
included in the multivariate analysis again. This time, age at surgery,
postoperative adjuvant chemotherapy and mTNM stage were found to be
independent prognostic factors for OS. The, biological risk and TNM
stage were not significant in the multivariate analysis (\textbf{Table
III} ).

\textbf{Predictive performance of m TNM staging system}

The differences in prognostic prediction between the eighth edition of
the TNM staging system and the mTNM classification system were compared
directly. The -2 log likelihood of the mTNM stage was 4688.361, which
was less than the value of the TNM staging system (4694.876), which
indicates a better performance in predicting survival (\textbf{Table V}
). The ROC curves showed that the AUC of the mTNM stage was
significantly greater than that of the TNM stage (AUC: 0.646 vs 0.587\%,
P=0.0007) (\textbf{Figure 6} ).

\textbf{Discussion}

Both tumor burden and malignant degree can reflect biological
characteristics of tumor, and they are also associated with the survival
of patients with malignancy. The AJCC TNM classification which reflects
tumor burden was the most widely used staging system for malignancy,
however, it was a macroscopic classification mainly based on anatomy and
did not incorporate the factors reflecting microscopic tumor burden
which could not be detected by imageology\textsuperscript{{[}14{]}},
neither the factors reflecting malignant degree of tumor. As a result,
there were always biases of accuracy when the TNM stage was used to
predict prognosis\textsuperscript{{[}8,9{]}}. In the present study, we
supposed to incorporate preoperative serum CA19-9, which could
compensate the deficiency of TNM stage in evaluating microscopical tumor
burden, and histology grate, which could make up for the defect of TNM
stage in reflecting malignant degree, into the TNM staging system to
reduce the biases.

In this study, patients with preopertive serum CA19-9 level
\textgreater{}112 U/ml demonstrated a significantly lower OS rate than
those with CA19-9 [?]112U/ml (5-year OS: 15.9\% vs. 22.6\%,
P\textless{}0.001). Patients with high grade also had a significant
decreased OS than those with low grade (5-year OS: 15.7\% vs. 23.5\%,
P\textless{}0.001). In the multivariate analysis, both preoperative
serum CA19-9 and histology grade were identified as independent
prognostic factors for OS. Our proposed biological risk of PDAC was
established according to the preoperative serum CA19-9 and histology
grade. All patients were categorized into three groups according to the
biological risk of PDAC: low-risk, middle-risk and high-risk group.
Significant differences in OS were observed among the three groups.
After incorporating the biological risk into the eighth edition of TNM
staging system, mTNM staging was established. It was confirmed that the
mTNM staging system was more accurate in predicting postoperative
prognosis of PDAC patients than the eighth edition of the AJCC TNM
staging system.

CA19-9 was the most widely used serum tumor marker. Kang et al reported
that both pretreatment and posttreatment CA19-9 levels or their changes
after treatment had good prognostic value in determining the survival of
pancreatic cancer patients\textsuperscript{{[}17{]}}. At present, there
was no recognized cut-off value of CA19-9 to predict the prognosis of
PDAC \textsuperscript{{[}18,19,20,21{]}}, as the level of CA19-9
fluctuated very widely, in our study, the CA19-9 level ranged from 0.6
to 178850 U/ml, besides, the proportion of negative Lewis-antigen
patients varied across countries, ethnicities and studies (6\% in white
population, 22\% in black population, 5-10\% in yellow
population)\textsuperscript{{[}22,23,24,25{]}}. Even so, multiple
researches have confirmed the advantage of CA19-9 over other serum tumor
markers in predicting prognosis of PDAC\textsuperscript{{[}26,27,28{]}},
which was consistent with our results.

Histology grade was defined as the degree of differentiation of the
tumor. Differentiation refers to the morphologic and functional
resemblance between a tumor cell and a normal cell of the same tissue.
Malignant neoplasms usually evolve from low grade to high grade. In the
process of malignancy, the higher the tumor grade, the more aggressive,
and the greater the tumor burden\textsuperscript{{[}29,30{]}}. Many
studies have demonstrated that histology grade was significantly
associated with survival of patients with PDAC and could be used to
modify the TNM staging system\textsuperscript{{[}31,32{]}}, so was the
case in our study.

To the best of our knowledge, this study is the first to categorize
patients with PDAC according to the biological risk based on
preoperative CA19-9 and histology grade, and assess the impact of tumor
biology on the survival. Our results reveled that patients with low risk
had a significant higher OS than those with middle risk, and patients
with middle risk also demonstrated a better prognosis than those with
high risk. The biological risk was confirmed to be an independent
prognostic factor in the multivariate analysis. In our opinion, the
combination of preoperative serum CA19-9 and histology grade contains
unique prognostic information which represents the microscopic tumor
burden and malignant degree, which is not included in the TNM staging
system.

There are several limitations to the study. First, it was a
retrospective study conducted at a single center. Second, patients were
included from 2011 to 2020, therefore, current neoadjuvant therapy were
not used routinely. In addition, we could not exclude patients with
negative Lewis antigen, which may have an uncertain impact on accuracy.
As the constraints listed above may cause bias in the results, data from
other centers is required for further validation of our findings.

\textbf{Conclusion}

All in all, preoperative serum CA19-9 and histology grade were
independent prognostic factors for patients with PDAC. Biological risk
which consists of preoperative serum CA19-9 and histology grade could
predict survival of patients with PDAC. Incorporating the biological
risk could improve the the accuracy of the TNM staging system in
predicting prognosis of PDAC.

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\textbf{Figure legends:}

\textbf{Figure 1} Receiver operating characteristic curve for predicting
survival of pancreatic cancer by various cut-off values of
CA19-9(AUC=0.576, 95\%CI:0.535-0.615, P=0.003). Cut-off value of CA19-9
with the maximum sensitivity + specificity for predicting survival of
pancreatic cancer was 112 U/mL.

\textbf{Figure 2} Overall survival curves after curative resection. (A)
Patients grouped according to CA19-9 level (P \textless{}0.001, log-rank
test). (B) Patients grouped according to histology grade (P
\textless{}0.001). Low grade: well and moderate differentiation, high
grade: poor differentiation and undifferentiation.

\textbf{Figure 3} Overall survival curves after curative resection. (A)
4 groups according to the status of CA19-9 and histology grade: group 1,
CA19-9\selectlanguage{english}[?]112 U/ml and low grade; group 2, CA19-9\selectlanguage{english}[?]112 U/ml and high grade;
group 3, CA19-9\textgreater{}112 U/ml and low grade; group 4,
CA19-9\textgreater{}112 U/ml and high grade. (B) 3 groups according to
the biological risk score: low risk group: CA19-9\selectlanguage{english}[?]112 U/ml and low
grade; middle risk group: CA19-9\selectlanguage{english}[?]112 U/ml+high grade or
CA19-9\textgreater{}112 U/ml+low grade; high risk group:
CA19-9\textgreater{}112 U/ml+high grade.

\textbf{Figure 4} Overall survival curves stratified by TNM stage.The
survival differences were only observed in pancreatic cancer patients
with stage I and stage II disease.(A) Stage I.(B) Stage II.(C) Stage
III.(D) Overall survival curves of pancreatic cancer patients according
to the TNM stage and biological risk.

\textbf{Figure 5} Overall survival curves of all pancreatic cancer
patients. There were significant differences in OS with mTNM or TNM
stage. (A) mTNM stage. (B) TNM stage.

\textbf{Figure 6} Receiver operating characteristic curve for comparing
the area under curves between mTNM and TNM. AUC(mTNM)=0.646, AUC(TNM)
=0.587,P=0.0007.

\textbf{Hosted file}

\verb`Figure.pptx`  available at \url{https://authorea.com/users/629147/articles/649503-biological-risk-based-on-preoperative-serum-ca19-9-and-histology-grade-predicts-prognosis-in-patients-with-pancreatic-cancer}

\textbf{Hosted file}

\verb`Table.docx`  available at \url{https://authorea.com/users/629147/articles/649503-biological-risk-based-on-preoperative-serum-ca19-9-and-histology-grade-predicts-prognosis-in-patients-with-pancreatic-cancer}

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