Evaluation of the effectiveness of serum aromatase activity and tissue polypeptide specific antigen in the diagnosis of breast cancer and prediction of lymph node metastatic status

Evaluation of the effectiveness of serum aromatase activity and tissue polypeptide specific antigen in the diagnosis of breast  cancer and prediction  of lymph node metastatic status

Taha I Hewala,a MD; Gehan M Shehata,b MD; Mahmoud A Hemida,c MD; Nadia A Abd El-Moneim,d MD

a) Departments of Radiation Sciences

b) Biomedical Informatics and Medical Statistics c)Experimental and Clinical Surgery

d)Cancer Management and Research

Medical Research Institute, Alexandria University, Egypt.

Co"espondence:e-mail: tahahewala@hotmail.com

Abstract

Aim: The aim of this study was to evaluate the effectiveness of serum tissue polypeptide specific antigen (TPS) and aromatase activity in the detection of primary breast cancer and prediction of lymph node metastasis.

Subjects and methods: Serum samples were obtained from 35 postmenopausal breast cancer females before surgery and 35 normal healthy volunteers. The clinicopathological  data of patients were obtained from their pathological reports. Determination of serum TPS was done using a ready-for-use ELISA kit. Serum total estradiol and total testosterone were determined using ready-for-use RIA kits and the ratio of estradiol to testosterone (E2/T) was used as an indirect expression of aromatase activity. Univariate and multivariate logistic regression analysis were performed to evaluate the association between these biomarkers and breast cancer, as well as lymph node metastasis.

Results: For breast cancer prediction, either serum TPS or aromatase activity can be used with significant areas under the ROC curves of97% & 91.8%, respectively. For lymph node metastasis prediction, serum aromatase activity alone had the best predictive value, with a significant area under the ROC curve of 80.3%. Multivariate logistic regression analysis in which aromatase activity and TPS were included in one model gives 74% adequacy in prediction of lymph node metastasis.

Conclusion:To predict breast cancer, either serum TPS or aromatase activity can be applied, while, for prediction of lymph node metastasis serum aromatase activity is recommended.

Key words:  Aromatase  activity,  tissue  polypeptide  specific  antigen,  breast  cancer.

Introduction:

Clinical examination, mammography, fine needle aspiration cytology and open surgical biopsy are established diagnostic methods in breast cancer and the fmal diagnosis of cancer should be based on histopathology.l Inbreast cancer, many tumor markers have been studied in the hope for finding a blood test for cancer diagnosis, but  none  of them  has  had  such sensitivity and specificity that it could replace conventional  diagnostic  methods.2

A number  of serum  markers  are used as

indicators for breast cancer. Of these, the most widely used are CA 15-3 and CEA. However, lack of sensitivity and specificity preclude the use  of  these  serum  markers for  the  early detection of breast cancer. There is an urgent need for cancer biomarkers with more accurate diagnostic  capability,  particularly for early­ stage cancer.3 Several clinical studies have been published on tissue polypeptide specific antigen (TPS) determination in women with breast cancer with sensitivity ranging from 31-

78%.4,5

Tissue polypeptide specific antigen (TPS) is related  closely  to human  cytokeratin 18, which  is an essential part of the protein cytoskeleton of epithelial cells.6 As opposed to CA 15-3,  which serum  concentration provides data about tumor mass, serum TPS levels indicate proliferative activity of malignant  tissue even if the mass is smalJ.7

Elevated concentrations of TPS have  been detected in association with a wide variety of malignancies, including breast cancer.s Human aromatase,  a  member  of  the cytochrome P450 superfamily, is a 58 kDa.It catalyzes  a complex  reaction  sequence  that results in the conversion of androgens (C19), namely testosterone and androstenedione, into estrogens (C18), namely estradiol, and estrone, respectively.9 In women of reproductive age, the ovaries express high levels of aromatase and they are the main  source  of estrogens. After menopause peripheral tissues including breast, lung, brain, and liver become sites of estrogen synthesis as a result of aromatization of  circulating  androgens.lO Aromatase expression is elevated in certain malignancies, such as breast and endometrial carcinomas.11

The aim of this study was the comparative evaluation of aromatase activity and  TPS concentrations in sera from patients with breast cancer  before  surgery and  normal  healthy controls. The diagnostic values of the assayed serum parameters were compared  using the Receiver Operating Characteristic (ROC) curve analysis. The correlation between biomarker levels and breast cancer histopathological characteristics was  determined. We  also investigated whether any of the biomarkers or their combined measurement could be used in the prediction of lymph  node involvement. Univariate survival analysis of patients was assessed using  the  Kaplan  Meier  method.

Subjects and  methods: Sample size calculation:

Sample size was calculated using  NCSS

2000 (Number  Cruncher Statistical System) and PASS (Power Analysis and Sample Size) Program. According to the results obtained by Hang&Rong-cheng for TPS,12 the minimum sample  size required was 40 (20 for breast cancer patients group and 20 for control group)

to achieve 80% power to detect a difference in means of 236 between both groups with estimated group  standard  deviations of 351 and 35 and with a significance  level (alpha) of 0.05  using  a two-sided sample  t-test.13

Seventy postmenopausal females were enrolled in this retrospective case- control study. A  female was considered postmenopausal if more than 12 months passed since her last menstrual period. Females were divided into two groups: Groupi (breast cancer patient's group):It included 35 female patients with breast invasive ductal carcinoma of clinical stages  II and III 14 (recently detected, not receiving surgery or chemotherapy). Their mean age was (51.33±2.12) years.  Patients were  recruited from  the  Departments of Experimental and Clinical Surgery and Cancer Management & Research of the Medical Research  Institute, Alexandria  University in the period from October 2007 to May 2008. Groupll (control group): It included 35 normal healthy volunteers of comparable age (52.80±3.50), menstrual cycle and socioeconomic status as patients.

This research protocol was approved by the ethical committee of  Medical Research Institute. Each patient underwent full history recording, thorough clinical examination, routine  laboratory investigations including complete blood count (CBC), mammography of breast and ultrasonography of abdomen and liver, radiological investigations including X­ ray chest, CT scan and bone scan when needed and fine needle aspiration cytology (FNAC) of breast mass to establish the pathological diagnosis in the patients.

Pathologic information was obtained from pathology reports. Collected data  included tumor size and grade, lymph node involvement and  status  of estrogen receptor (ER)  and progesterone receptor (PR). Breast cancer was clinicaly  staged by the oncologist  according to the  tumor-nodes-metastasis (TNM) classification.l4

All 35 breast cancer patients were subjected to surgery (Modified Radical Mastectomy),15 then received adjuvant combination chemotherapy [5-Fluorouracil, Adriamycin and Cyclophosphamide (FAC)] 16 for 6 cycles. Breast cancer patients were evaluated after 3

and 6 cycles  of chemotherapy clinically, laboratory and radiologically to estimate the clinical response. Also, these patients were followed up till May 2011 for assessment of overall survival.

Laboratory investigations:

Blood samples were collected from normal

healthy volunteers and breast cancer patients before surgery. Immediately after withdrawing, blood samples were allowed to coagulate and were centrifuged for 20 minutes at 3500 rpm. The separated serum samples were aliquoted, frozen at -80 °C, and stored until assay time. After thawing, each serum aliquot was assayed only once. Determination of serum levels of tissue polypeptide specific antigen (TPS), total testosterone and estradiol were carried out at Radiation Sciences  Department, Medical Research  Institute,  Alexandria  University.

Determination of serum Tissue Polypeptide

Specific Antigen (TPS):

Serum TPS levels were determined using a ready-for-use ELISA kit (DRG diagnostics, Germany) according to the manufacturer's protocol. Briefly, 50 J.Ll of each calibrator and patient's serum sample were added into each well. 50 J.Ll  of the diluted HRP-conjugated Anti-TPS solution to each well. Wells were shaken on a rack shaker (600 rpm) for 2 hrs at room temperature. Wells were decanted and washed with 6 x 0.3 m1diluted wash solution.

200 J.Ll tetramethyl-benzidine (TMB) substrate

solution were added per well and incubated in darkness for 20 min. 50 J.Ll  of stop solution were added per well and the wells were mixed well. Absorbance of every well was read at

450 nm.  A standard curve was constructed

from which the concentration of TPS in each serum sample was determined. The assay sensitivity was 6 U/L with no detectable cross reactivity to other tumor associated antigens that may be present in serum.

Determination of serum total testosterone: Serum  total  testosterone levels  were determined using a ready-for-use solid- phase radioimmunoassay kit (RIA, Siemens, USA) according  to the  manufacturer's protocol. Briefly, 50 J.Ll of each calibrator and patient's

serum sample were pipetted into the test tubes.l.O ml of t25I-labeled total testosterone tracer was added to every tube. Tubes were mixed well and incubated for 3 hrs at 37 °C, and then tubes were decanted thoroughly. Bound radioactivity in each tube was counted for 1 minute in a gamma counter (Perkin Elmer, Finland). A calibration curve was constructed from  which  the  concentration of total testosterone (ng/dl) in each serum sample was determined. The assay sensitivity was 4 ng/dl with  high   specificity  for  testosterone.

Determination of serum  total  estradiol: Serum total estradiol levels were determined using  a   ready-for-use   solid-  phase radioimmunoassay kit (RIA, Siemens, USA) according  to the  manufacturer's protocol. Briefly, 100 J.Ll of each calibrator and patienf s serum sample were added into the test tubes.

1.0 m1of125I-labeled estradiol tracer was added to every tube. Tubes were mixed well and incubated for 3 hrs at room temperature, and then tubes were decanted thoroughly. Bound radioactivity in each tube was counted for 1

minute in a gamma counter (Perkin Elmer,

Finland). A standard curve was constructed from which the concentration of estradiol (pg/ml) in each serum sample was determined The assay sensitivity was 8 pg/ml with high specificity for estradiol.

Estimation of serum  aromatase activity: Each serum value of estradiol (pg/ml) was multiplied  by 10 to be converted into its corresponding ng/dl unit. According to several studies, aromatase activity in each serum sample from every patient and normal healthy volunteer was estimated indirectly as the ratio of the total estradiol (ng/dl) level divided by total testosterone  level (ng/dl); (E2/T).17-19

Statistical analysis:

A- Data processing:

Data processing had two major objectives namely clean data by performing a series of comprehensive checks and producing analytic results  which involved the recoding of variables.

i. Recoding of variables:  The quantitative

variables were recoded using different cut

off  points  defined by ROC  analysis to include them in the analysis.

ii.  Exploration of the quantitative data  for normality distribution.

B- Data analysis included:

1- Kolmogrov- Smirnov test  for  data exploration.

2- Calculation of the median and range for the

quantitative variables.

3-  Mann  Whitney test  was  performed to compare between the medians of the two groups.

4- Logistic regression analysis is used to assess

the impact  of interaction between two predictors  as TPS and aromatase on the lymph node involvement in breast cancer patients.

5- ROC curve analysis was done to detect the area under the curve (overall accuracy) and different cut off points with the associated sensitivity and specificity.

6-  Survival analysis was  done  including

survival life tables, Kaplan Meier's curves and Log Rank test to compare between two factors regarding the survival. P value less than  0.05  was  considered statistically significant.

Results:

1- The diagnostic value of semm TPS  and

aromatase activity:

Serum  TPS level and aromatase activity were significantly elevated in the breast cancer patients group compared with the control group Table(l). Comparing the diagnostic values of serum TPS and aromatase activity  using the receiver operating characteristic (ROC) curve analysis showed significant greater area under the curve for TPS (97%) compared with that for aromatase activity (91.8%). The sensitivity and specificity were 97.1% and 85.7% at cut­ off 67.5 U/L for TPS and 86% and 89% at cut­ off 0.09 for aromatase activity Figure(l) and Table(2).

Table (1): TPS and aromatase activityin the breast cancer patients and normal healthy control groups.

*:Significance was considered at P-value < 0.05 n:   Number  of   subjects  in   each  group

1.25

1.00

.:.•n..................-.......

...._. ...,._                      Aromatase activity

•

.75 ,.!

.:-)o4

o.oo._  

----  ----  -----4

0.00

.25

.50            .75

1.00

1 - Specificity

Figure(1): Graphical representationof the ROC curves for serum TPS and aromatase activity for detection of breast cancer.

Table (2): Area under the curve (AUC),cut-off, sensitivity and specificity for serum TPS

(U/L) and aromlltaseactivity fordetectionof breast cancer.

*:Significance was considered at P-value < 0.05

IT- Correlation of serum TPS and aromatase activity with  breast           cancer clinicopathological characteristics: Serum TPS has no correlation with any of

the clinicopathological characteristics ofbreast cancer patients. Wbilet aromatase activity was significantly correlated with  lymph  node involvement and tumor clinical stage Table(3). Comparison of the power of each serum marker to predict lymph node metastatic status using ROC curve yield a significant greater AUC for aromatase activity (80.3%) than that of TPS (38.9 %). Also, from ROC

curves,  the  sensitivity and  specificity of aromatase activity were 76.2% & 85.8% at cut-off 0.155, while  the  sensitivity and specificity ofTPS were 71.4% & 21.5% at cut-off  92.5 U/L Figure(l) and Table(4). Multivariate logistic regression analysis in which  TPS  and  aromatase activity were included in one model gives AUC of 74%.

Relationship between  serum TPS and

aromatase activity and  patients' overall survival:

Kaplan-Meier analysis revealed that patients

with  elevated levels  of  serum  TPS  and

aromatase activity  had   a  survival non­

significantly shorter than those who had lower

levels of  these   biomarkers as  shown in

Table(S).

Table  (3): Serum levels  of  TPS  and  aromatase activity [median (range)] in relation to clinicopathological characteristics of breast cancer patients.

*:Significance was considered at P-value < 0.05

ER & PR: Estrogen  and progesterone receptor

l.OO ..----------------.

Aromatase activity   1

.75

. 50  -

.-- )

J

r--

1

,J

.25

....

IJ TPS

_.JJ

0.00      --      --             -         - --£

0.00          .25           .50                  .75           L OO

1 - Specificity

Figure (2): Graphical representation of the ROC curves for serum TPS (U/1) and aromatase activity  for prediction  of lymph  node involvement  in  breast  cancer  patients  group.

Table (4): Area under the curve (AUC), cut-off, sensitivity and specificity for serum TPS (UIL) tllld t romatase tiCtivity for predicdon of lymph node involvement in breast cancer patients group.

*:Significance was considered at P-value < 0.05

Table (5): Correlation between serum TPS levels, aromatase activity and overall survival

(months) among breast cancer patients group.

Discussion:

Early detection ofbreast cancer can improve cancer mortality, and early prediction of lymph node metastasis  may facilitate  the choice  of operation type  as  well  as use  of adjuvant therapy. The use of serum markers has been well established in the screening and post­ operative follow-up of different types  of malignancies.20 The present most commonly used breast cancer serum markers, carcinoembryonic antigen (CEA) and breast cancer-specific cancer antigen 15.3 (CAl 5-3), have disadvantages including poor sensitivity, poor specificity and a lack of standard cut-off values for either primary breast cancer detection or  metastatic status prediction.21 Several biomarkers have been proposed as potential new markers for breast cancer, including tissue polypeptide-specific antigen (TPS)  which measures a specific epitope structure of soluble fragments ofhuman cytokeratin 18, an acidic­ type intermediate filament protein detected in various types ofhuman cancer.6

For  TPS,  at a cut-off level  of  75-170

U L-1, the  sensitivity and  specificity for detection of primary breast cancer were reported to be 30-95% and 75-90%, respectively.22,23 Kassim et al used TPS as a sensitive proliferative serum marker in screening for  those  at high  risk  of  breast cancer.22 Hwa et al. showed that TPS was the

best predictive marker for primary breast cancer at a cut- off value of 69.1 U/L with a sensitivity of 80% and specificity of 67%.21

The findings of this study support  serum TPS  to be  a diagnostic serum  marker  that differentiates breast cancer  patients from normal healthy controls. At cut-off value of

67.5 U/L, TPS has sensitivity and specificity of97.1% & 85.7%, respectively, with AUC of 97%. Our results  are in agreement with Kassim et al.22 who  used  serum TPS  to differentiate Egyptian  breast cancer females from females with bengin breast diseases.They reported that at a cut-off for TPS of 88 U/L, the sensitivity is 95%, and the specificity  is

75% with AUC of 91%. The greater cut-off value in Kassim et al. study 22 may be due to the use of patients with benign breast diseases instead  of normal  healthy  volunteers. Also, different distribution of clinical stages of studied cases may   influence the  cut-off  value.

Concerning the diagnostic value of serum aromatase activity, the present study showed the  possibility of  using this  enzyme to differentiate between  breast cancer females and normal healthy volunteers. At cut-off value of0.09, aromatase activity has sensitivity and specificity of 86 % & 89%, respectively, with AUC of91.8%. By comparing the diagnostic values of serum TPS and aromatase  activity using ROC curve, the AUC of TPS was found

to be significantly greater thanthat of aromatase activity indicating that  TPS  is superior to aromatase activity in detecting breast cancer in postmenopausal females. To the best of our knowledge, no other previous or recent work studied the diagnostic  role of this enzyme in the sera ofbreast cancer females.

The present study reveals that serum TPS

is correlated with neither clinicopathological characteristics nor overall survival of breast cancer patients. Our study is compatible with the results reported by Hu et al.24

In our study, serum aromatase activity was significantly correlated with  lymph node metastatic status and patient clinical stage. On the  other  hand,  aromatase activity has  no significant correlation with patients' overall survival. The  absence of a significant correlation between  serum  TPS  levels  and aromatase activity with patients overall survival may be related to the small sample size used in the present study.

Univariate logistic regression analysis for prediction of lymph node metastasis  showed that  serum  aromatase activity has the best predictive value  for lymph  node metastasis with  AUC  of  80.3%, the  sensitivity and specificity were 76%, 85.8 %, respectively, at a cut-off0.155.

The multivariate logistic regression model

is a simple method  for considering  multiple continuous and/or categorical factors simultaneously. It can calculate the weighting on  the individual prediction. A variety of markers, each of which had been reported to be elevated in patients with cancer of any type, were combined to see if multivariate analysis would yield information not available by the use of one marker alone.21 The developed multivariate logistic regression equation used in the present  study  presents the degree  of contribution of each variable to the prediction.

To  study  the  effect of  combination of aromatase activity and TPS on the prediction of lymph node metastatic status, multivariate logistic regression analysis showed that combination of serum  levels  of aromatase activity and TPS resulted in 74% adequacy of predicting lymph node metastasis which means that  this  combination did not  improve  the accuracy of predicting lymph node metastasis

achieved by  aromatase activity alone. Therefore, to screen for breast cancer, either serum TPS or aromatase activity can be used, but  in  case  of  screening for  lymph  node metastasis in breast  cancer  patients, serum aromatase activity can be assayed with no need for assaying TPS in order to reduce the patient cost. This can meet the demands of different populations of different sizes.

In conclusion, serum  TPS  or aromatase activity  can be used for detection  of breast cancer, while only serum aromatase activity can be used as a predictive marker for lymph node metastasis.

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