plaque brachytherapy, external beam radiotherapy and enucleation according to the ... the chemotherapy cycles and / or external beam radiotherapy then every.
Title: Expression of transforming growth factor beta-1 protein in aqueous humor and serum of retinoblastoma patients and its clinical significance
Name of authors: 1
2
3
Azza Mohamed Ahmed Said MD. Saffaa Saleh Mohamed MD. Hanan Hussein Shehata MD. Azza 4
Hassan Abou Ghalia MD. , Eman Khairy Elsayed M.Sc.
5
1- Lecturer of Ophthalmology, Faculty of Medicine, Ain Shams University. 2- Assistant professor of Ophthalmology, Faculty of Medicine, Ain Shams University. 3- Lecturer of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Ain Shams University. 4- Professor of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Ain Shams University. 5- Assistant lecturer of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Ain Shams University.
Abstract Purpose: Quantitative measurement of transforming growth factor beta-1 (TGF-β1) protein in both aqueous humor and serum of retinoblastoma children and its clinical relevance. Subjects and Methods: A case-control study was carried out in Ophthalmology and the Medical Biochemistry departments, Ain Shams University in the period from March 2007 to July 2009. The study included (20) newly diagnosed children with retinoblastoma in whom enucleation was indicated in at least one eye and (20) age matched children with ophthalmic conditions rather than tumors prepared for intraocular surgeries, served as controls. Twelve patients (12) who underwent enucleation (part of the 20 preoperative retinoblastoma patients) also were included as a follow up group. They were selected randomly from patients who underwent surgery. Histopathological analysis of the enucleated eyes was performed. Venous blood samples (2 mL) and aqueous humor samples (100–150 μL) had been obtained from all children to measure TGF-β1 concentration. Results: The mean concentration of TGF - 1 in serum and aqueous samples from patients with retinoblastoma were statistically significantly higher than the corresponding values in the control group (P values ≤ 0.01). The mean concentration of serum TGF-1 in patients with retinoblastoma before treatment was 433.14 (pg/ml) ± 56.29 SD. After treatment, it was 365.92 (pg/ml) ± 62.3 SD. This reduction was highly statistically significant (P value =. 013). In the control group; it was 320.8 (pg/ml) ± 83.19 SD. The mean concentration of aqueous TGF-1
in patients with retinoblastoma before
treatment was 87.59 pg/mg protein ±37.98 SD and in the control group it was 23.06 pg/mg protein ±11.49 SD. This difference was highly statistically significant (P≤ 0.01). The best cutoff values of serum and aqueous TGF- 1 which gave the highest sensitivity and specificity were 349.37 pg/ml protein and 39.82 pg/mg protein respectively. All retinoblastoma patients had serum and aqueous TGF-1 above the cutoff values. Meanwhile, there was highly statistically significant positive correlation between aqueous TGF- 1 and differentiation of the tumor (P≤ 0.01).
Conclusion: Increased expression of TGF-1 in serum and aqueous humor of retinoblastoma patients with high sensitivity and specificity and significant correlation with degree of differentiation of the tumor might be used as a molecular marker for the diagnosis as well as completeness of treatment of retinoblastoma.
Also, it indicated a promising future therapeutic strategy based on its inhibition.
Further studies with a large number of patients for a longer follow-up period are necessary. Keywords: Retinoblastoma, Transforming growth factor beta-1, Aqueous humor
Introduction In many cancer cases, tumor cells develop when normal progenitor cells lose their soluble growth-regulatory factors that inhibit cell cycle growth. Transforming growth factor beta (TGF- β) is the most commonly studied and the predominant form found in human; the TGF-β 1 has been shown to be a potent inhibitor of the proliferation of most normal cell types in culture as well as in vivo.
(1)
TGF-β is pleiotropic growth factor that regulates cellular growth and differentiation, apoptosis, cell motility, extracellular matrix production, angiogenesis and cellular immune response.
(2)
It is a
potent cytokine that modulates embryonic development, bone formation, mammary development, wound healing, and hematopoiesis.
(3)
This molecule is produced by a great number of cells and tissue
types including thrombocytes, bone tissue, placenta and kidneys.
(4)
Thus, TGF-β is a potent anticancer agent that prohibits the uncontrolled proliferation of epithelial, endothelial, and hematopoietic cells. TGF-β can induce apoptosis (programmed cell death) in numerous cell types through the Smad pathway (family of eight proteins) mediate TGF- β signaling inside the cells.
(1)
Interestingly, tumorigenesis typically elicits aberrations in the TGF-β signaling
pathway that engenders resistance to the cytostatic activities of TGF-β, thereby enhancing the development and progression of human malignances. Moreover, these genetic and epigenetic events conspire to convert TGF-β from a suppressor of tumor formation to a promoter of their growth, invasion, and metastasis. The dichotomous nature of TGF-β during tumorigenesis is known as the “TGF-β Paradox,” which remains the most critical and mysterious question concerning the physiopathological role of this multifunctional cytokine. Kelly and Morris
(2)
(5, 6)
reported that TGF- β facilitates malignant transformation and stimulate
tumor growth by manipulating a more hospitable environment for tumor invasion and the development of metastasis. Mechanisms that may explain these paradoxical activities are diverse. They also demonstrated that altered TGF-β signaling in the form of the inactivation of various components of the TGF-β receptor signaling system, including mutations of TGF-β receptor II (TβRII), TβRI, and the various Smad proteins lead to decrease sensitivity to the inhibitory effects of TGF-β. Increased TGF-β expression and its activation leading to epithelial-to-mesenchymal transition, increased invasiveness, metastasis, angiogenesis and immunosuppression.
However, most types of cancer do not harbour TGF- β mutations, suggesting that TGF- β signaling could be functional in those cells. It seems that functional interaction with other pathways can promote initial resistance to the inhibitory activity of TGF- β.
(7)
TGF-β is over expressed by many
tumors including cancers of the breast, colon, esophagus, stomach, liver, lung, kidney, pancreas, prostate, brain, and malignant melanoma, as well as certain hematological malignancies. Over expression of TGF- β has been correlated with advanced tumor stage, metastases and a poorer overall prognosis.
(8-13)
However, little is known regarding aqueous and serum levels of TGF- β1 in patients with retinoblastoma and its clinical relevance with tumor treatment. The aim of this study was quantitative measurement of TGF-β1 protein in both aqueous humor and serum of retinoblastoma children and finding a correlation of this cytokine levels with the clinicopathological criteria.
Subjects and Methods Study population A case-control study was carried out in Ophthalmology and the Medical Biochemistry departments, Ain Shams University in the period from March 2007 to July 2009. The study included (20) newly diagnosed children with retinoblastoma in whom enucleation was indicated in at least one eye and (20) age matched children with ophthalmic conditions rather than tumors prepared for intraocular surgeries, served as controls. Twelve patients (12) who underwent enucleation (part of the 20 preoperative retinoblastoma patients) also were included as a follow up group. They were selected randomly from patients who underwent surgery. Informed consent had been taken from parent of each participating subject. All children in the study have had careful history taking.
Complete ophthalmological
examination was conducted under general anesthesia with full pupillary dilatation for the retinoblastoma group. Fundus examination using the indirect ophthalmoscope with scleral indentation was performed. Fundus photography using fundus camera (GENESIS D, KOWA MEDICALS, JAPAN) was made and the clinical findings were documented. According to Reese – Ellsworth classification (14)
, retinoblastoma staging was recorded. B-scan ultrasonography was performed to determine tumor dimensions and to confirm the
presence of intraocular calcification. Computed tomography of the orbits and brain was conducted to detect any intracranial extension and trilateral retinoblastoma. A magnetic resonance imaging was not obtained as a routine method of examination except in cases with no visualization of the optic nerve head during clinical examination. Retinoblastoma children received one or combination of the following treatment modalities; chemo reduction with focal consolidation therapies (cryotherapy and laser thermotherapy), episcleral plaque brachytherapy, external beam radiotherapy and enucleation according to the retinoblastoma staging. Diode laser thermotherapy has been used to treat small tumors posteriorly located in Reese group (I) or (II) using (IRIDEX OcuLight SLx Infrared (810 nm) laser system).
Treatments were
administered in the form of monthly cycles with chemotherapy was given on the first 2-3 days of each month. Laser was set at continuous mode and the power was adjusted at 300-400 mw.
Triple freeze-thaw cryotherapy was the primary treatment of single or multiple pre-equatorial tumors 6 mm or less in diameter. Systemic chemotherapy using a triple drug protocol consisting of 2
2
2
Vincristine (1.5mg/ m ), Etoposide (200 mg/m ) and Carboplatin (560 mg/m ) was given as first line and an adjuvant treatment to the previous modalities. This was administrated in 6-12 cycles. EBRT was given for large tumors more than 13 mm in diameter, tumors with vitreous seeding and tumors of any size near the optic disc. Histopathological analysis of the enucleated eyes was performed. Ophthalmological follow up of children to detect tumor regression and presence of resistance of the tumor to treatment was done every month till finishing the chemotherapy cycles and / or external beam radiotherapy then every three months for the first year then at longer intervals according to the tumor control. Sample collection and storage Venous blood samples (2 mL) had been taken using disposable syringes from 20 retinoblastoma children in the preoperative period and six months after the last treatment had been given in 12 patients. In addition, sera from 20 age matched children had been collected in the same fashion and used as a control at the time of analysis. Sera had been obtained after centrifugation of clotted blood samples. Samples had been divided into aliquots, and then stored at−20 °C till laboratory analysis. Aqueous humor samples (100–150 μL) had been obtained from children in both groups under complete aseptic conditions in the operating theatre. All children had general anesthesia. In the retinoblastoma group who were candidate for enucleation, the samples were taken after transection of the optic nerve. Anterior chamber paracentesis was done using 27 gauge disposable syringes with aspiration of aqueous humor using the same syringe. In the control group samples were taken at the start of the surgical procedure through anterior chamber paracentesis. Samples were then divided into aliquots and stored at −20 °C till laboratory analysis. Care was taken to avoid specimens be contaminated with blood. Assay procedures TGF-β1 concentration was measured in serum and aqueous humor samples using The DRG® TGF-β1 ELISA KIT is a solid phase enzyme-linked Immunosorbent Assay (ELISA), which is based on the sandwich principle. Prior to testing, the standards and patient samples were diluted in assay
buffer, acidified with HCl and then neutralized with NaOH. Afterwards, the neutralized standards and samples were added to the antibody coated microtiter wells. After the first incubation, the unbound sample material was removed by washing with diluted wash solution. Then a monoclonal mouse anti TGF-β1 antibody, a biotinylated anti mouse IgG antibody and the streptavidin-HRP Enzyme complex were incubated in succession. An immunoenzyme sandwich complex was formed. The unbound conjugate was removed by washing. Subsequently, substrate solution was added. After a definite time, color development was stopped by addition of stop solution and the absorbance at 450 nm was measured with a microtiterplate reader. The intensity of the color development was proportional to the TGF-β1 concentration in the sample.
(15)
The total protein concentration in aqueous humor samples was determined according to Bradford
(16)
TGF-1 concentration was measured in serum and aqueous humor samples from the three groups. Statistical analysis Data were analyzed using SPSS 13 software. Student's t test was performed to assess whether the results are significant or not. The level of significance was set at P ≤ 0.05. One-way analysis of variance (ANOVA) was used to test the significance between more than two groups. Pearson's correlation coefficient (r) was performed to test the correlation between TGF-1 expression and different variables. The best cutoff value that maximizes sensitivity and specificity and differentiates retinoblastoma patients from controls was calculated by using the ROC (receiver operating characteristic) curve.
Results Eleven (11) females (55%) and nine males (45%) of newly diagnosed retinoblastoma children were included in the study group. The mean age of this group was 18.6 months ±13.8 SD (range; 2-60 months). Unilateral retinoblastoma was the diagnosis in nine (45%) patients and affection of both eyes in 11 (55%) patients. Leucocoria was the presenting sign in 17 patients (85%) and squint in three patients (15%). Positive family history was reported in two patients (10%) and first degree consanguinity in three patients (15%). Reese – Ellsworth classification was as follows in unilateral cases; two eyes (22.2%), one eye (11%), three eyes (33.3%) and the last three eyes (33.3%) were in group (II), (III), (IV) and (V) respectively. Regarding staging of the more advanced eyes in bilateral cases; one eye (8%) was in group (IV) and 10 eyes (91%) were in group (V). However the staging of the less advanced eyes in bilateral cases was as follows; four eyes (36.4%), six eyes (54.5%), one eye (9.1%)
were in group
(I), (II) and group (III) respectively. In unilateral cases; enucleation was the primary treatment of seven retinoblastoma eyes (77.7%) and it was secondary to failure of systemic chemotherapy and external beam radiotherapy in two eyes (22.2%). In the bilateral cases; enucleation was performed for all eyes with more advanced stage with prior two courses of systemic chemotherapy in two eyes. So the total number of enucleated eyes was (20 eyes).
The less advanced eyes in bilateral cases were treated by; Transpupillary
thermotherapy in two eyes (18.2%) , systemic chemotherapy in one eye (9%), systemic chemotherapy with focal laser therapy in six eyes (54.5%) and systemic chemotherapy and focal laser therapy followed by external beam radiotherapy in two eyes (18.2%). No evidence of recurrence in all cases along the follow up period. Histopathological examination was done of the enucleated eyes and revealed the following; poorly differentiated tumor in eight eyes (40%) and well differentiated tumor in 12 eyes (60%). Optic nerve infiltration was reported in two eyes (10%). The second group was the control group (20 patients) in which; the mean age was 14.75 months ± 14.5 SD (rang; 1- 48 months). This group included 11 males (55%) and nine females (45%). The indications for intraocular surgery were; congenital cataract in 12 patients (60%), secondary IOL implantation in five patients (25%) and congenital glaucoma in three patients (15%).
The third group was the follow up group which included 12 cases from the first group following treatment. The mean age was 33 months ± 2.7 SD (range; 14 – 78 months). The number of males and females was eight (67%) and four (33%), respectively. There was no statistically significant difference between the three groups as regards age and sex distribution. (P-values were 0.39 and 0.2 respectively) The mean concentration of TGF - 1 in serum and aqueous samples from patients with retinoblastoma were significantly higher than the corresponding values in the control group. Using the analysis of variance (ANOVA), the
mean concentration of serum TGF- 1
was
statistically
significantly higher in the malignant and follow up groups compared to control group, P values were ≤ 0.01 (Table 1). The mean concentration of serum TGF-1
in patients with retinoblastoma before treatment
was 433.14 (pg/ml) ± 56.29 SD and after treatment, it was 365.92 (pg/ml) ± 62.3 SD. So the serum level of TGF- 1 in the follow up group was decreased after treatment of retinoblastoma and this reduction was statistically highly significant when comparison was done between the concentration before and after treatment in the retinoblastoma group. (P value =. 013)
Table (1): The mean concentration of aqueous and serum TGF- 1
in patients with retinoblastoma
before and after enucleation (serum only) and the control group. Group
Aqueous TGF-1 ( pg/mg protein)
P-value
Mean ± SD ( Range ) Retinoblastoma
Control
Follow up
Serum TGF-1 (pg/ml) Mean ± SD ( Range )
87.59±37.98
433.14 ± 56.29
(40.5-166.8)
(351.9-519.8)
23.06±11.49
≤0.01
320.8 ± 83.19
(11.2-39.1)
(186.3-518.4)
Not applicable
365.9 ± 62.31
P-value
≤0.01
Regarding (ROC) curve and cutoff value of aqueous TGF- 1; the area under the ROC curve of aqueous TGF- 1
level (0. 985) and P value (0.000) are shown in (Fig. 1) and represented by
green line. The best cutoff value of aqueous humor TGF- 1 which gave the highest sensitivity and specificity was 39.82 pg/mg proteins. Applying this cutoff value, the maximum sensitivity, specificity, positive, negative predictive values and accuracy of aqueous humor TGF- 1 were 100%, 93%, 95%, 100% and 97%, respectively. The (ROC) curve and cutoff value of serum TGF- 1; the area under the ROC curve of serum TGF- 1 (0.87) and P value (0.000) are shown in (Fig. 2) and represented by green line. The best cutoff value of serum TGF- 1 which gave the highest sensitivity and specificity was 349.37 pg/ml. Applying this cutoff value, the maximum sensitivity, specificity, positive, negative predictive values and accuracy of serum TGF- 1 were 100%, 70%, 77%, 100% and 85%, respectively.
Aqueous TGF-1 Reference line
Figure 1: Receiver Operating Characteristic (ROC) curve showing the diagnostic performance of aqueous humor TGF-1. A black arrow points to cutoff value of 39.82 pg/mg proteins of aqueous humor TGF- 1 which gave a sensitivity of 100% and specificity of 93%.
Serum TGF- 1 Reference line
Figure 2: Receiver operating characteristic (ROC) curve showing the diagnostic performance of serum TGF-1. A black arrow points to cutoff value of 349.37 pg/ml of serum TGF- 1 which gave a sensitivity of 100% and specificity of 70%.
Using Chi square analysis, the percentage of patients with serum and aqueous TGF- 1 above the cutoff values were significantly higher (P 0.01) in the retinoblastoma group before treatment than the other two groups. (Table 2) All retinoblastoma patients had aqueous and serum TGF-1 above the cutoff values (39.82 pg/mg protein, 349.4 pg/ml respectively). Comparison was done between each of serum and aqueous TGF- 1 and all other clinico pathological factors (Table 3) and revealed a highly statistically significant difference was found between aqueous humor TGF- 1 and the degree of differentiation of the tumor (P-value was 0.003) however no statistically significant difference between them and progress in age, difference in sex, whether positive or negative family history of the disease and advanced stage of the disease. (Pvalues >0.05) Pearson’s correlation coefficient was done between each of serum and aqueous TGF- 1 and different variables: age, sex, family history, tumor classification, degree of differentiation of the tumor and presence of optic nerve affection
and revealed only
highly statistically significant positive
correlation between aqueous TGF- 1 and grade of differentiation of tumor (r = 0.732, P-value was 0.001).
Table (2): Comparison between the percentage of patients with aqueous humor and serum TGF- 1 above or below the cutoff value among the three studied groups Serum TGF - 1
Retinoblastoma (n=20 )
Control (n=20)
Follow up (n=12)
Above the cutoff (>349.37 pg/ml)
20 (100%)
6 (30%)
7 (58.3%)
Below the cutoff (< 349.37 pg/ml)
0
14 (70%)
Above the cutoff (>39.82 pg/mg protein)
20 (100%)
1 (5%)
Below the cutoff (< 39.82 pg/mg protein)
0
Statistical Significance
5 (41.7%)
X²: 21.309 P=.000
Not applicable
X²: 29.179 P =.000
Aqueous TGF- 1
19 (95%)
Aqueous humor and serum TGF- 1 levels in relation to different clinicopathological
Table (3):
factors in patients with retinoblastoma who were treated by enucleation Aqueous TGF- 1
Age 1-12 months (9) 13-24 months (7) >24 months (4) Sex Male (9) Female (11) Family history Positive (2) negative (18) Classification Group II-III (3) Group IV-V (17)
Serum TGF- 1
Pvalue
Median
Range
39.25 56.97 41.37
20.27-72.0 55.07-58.88 21.37-83.43
0.42 (NS)
Age 1-12 months (9) 13-24months (7) >24 months (4)
40.21 50.23
20.58-72.00 20.27-83.43
0.34 (NS)
Sex Male (9) Female (11)
24.7 46.92
20.27-29.13 20.58-83.43
0.92 (NS)
Family history Positive (2) negative (8 )
29.13 45.87
20.58-55.79 20.27-83.43
0.30 (NS)
Classification Group II-III (3) Group IV-V (17)
Grade Grade Well Well differentiated 0.003 differentiated 27.69 20.27-56.07 (12) (HS) (12) 57.33 40.21-83.43 Poorly Poorly differentiated (8) differentiated (8) Optic nerve Optic nerve Not affected 44.20 20.27-72.0 0.78 Not affected (18) 52.37 21.31-83.43 (NS) (18) Affected (2) Affected (2) NS; statistically non significant, HS; statistically highly significant
Pvalue
Median
Range
415.3 471.5 453.1
351.9-510.1 392.3-506.4 395.1-519.8
0.49 (NS)
471.5 402.96
351.9-519.8 372.1-471.5
0.234 (NS)
437.4 416.9
396.5-478.4 351.9-519.9
478.4 402.9
415.3-502.7 351.9-519.8
430.0 403.1
364.3-510.1 351.9-519.8
428.9 411.0
351.9-519.8 403.3-418.6
0.9 (NS)
0.126 (NS)
0.46 (NS)
0.90 (NS)
Discussion TGF-β is a cytokine involved in a wide variety of physiological and pathological processes. Regarding cancer, TGF-β initially contributes to maintenance of normal tissue and cellular homeostasis; nevertheless, at a particular time, TGF-β can’t suppress cancer development and, even more so, it acts as a promoter of tumoral progression, being secreted in large quantities by neoplastic cells of several cancer types. The “Dr Jekyll and Mr Hyde”- like paradox of TGF-β can be explained, at least in part, by the different composition of the stromal and microenvironmental elements.
(17)
Several
large and small drug molecules inhibiting TGF-β are being tested in preclinical and clinical trials.
(18, 19)
Considering retinoblastoma, the current study revealed significant increase in serum TGF-β1 in patients with retinoblastoma than in the control group (P
