Pathological Risk Factor Profile for Enucleated Retinoblastoma at Sanglah General Hospital

Retinoblastoma is a primary intraocular tumor on childhood. It has a good prognosis with early detection. Aims: Aim of this study is to know Pathological Risk Factors (PRF) profile of enucleated retinoblastoma at Sanglah General Hospital. Methods: This study is an observational retrospective study based on patient’s medical record and pathological examination. Twenty patients were included in this study, 50% were female and 50% male within age ranging from 7 month – 12 years old. Unilateral retinoblastoma was found in 18 patients (90.5%), bilateral retinoblastoma was found in 2 patients (9.5%). Majority PRF profile found that cancer cell invasions was 60% choroid infiltration and 45% of anterior segment invasion, 60% of optic nerve infiltration, and 50.1% scleral invasion. Tumor cell aggregation showed 61.9% poorly differentiated malignant cells. None of the patients had family history of retinoblastoma. This study revealed optic nerve and choroid invasion were the major PRF found in retinoblastoma, but there is a significant association between scleral infiltration and tumor cell differentiation (p = 0.025). There was no statistical difference of PRF based on age, gender, and laterality.

Retinoblastoma is a primary intraocular malignancy that occurs most often in children with incidence of 1:20.000.Developed under 5 years old children, it mostly occurs in the first two years of life. 1,2,3The incidence on both sexes are almost the same.Retinoblastoma patient life expectancy exceeds 95% in western countries and decreased to less than 50% in case of the extraocular spreading. 2,4athological Risk Factors (PRF) of the tumor is the result of eye enucleation pathology and associated with a higher risk of extraocular relapse. 5,6For a hundred years, we have been observing two typical true rosettes in retinoblastoma in the form of Flexner-Wintersteiner (FW) and Homer Wright (HW) rosettes. 7Pathological Risk Factors include the invasion of the anterior segment, minor choroid invasion, invasion of major choroid, pre lamina optic nerve invasion, post lamina optic nerve invasion, intra sclera invasion and trans sclera invasion.High-risk Pathological Risk Factor (HRF) includes major invasion of the choroid, pre and post lamina optic nerve invasion, and intra-trans sclera invasion. 7,8n Indonesia and other developing countries, diagnosis of retinoblastoma had only done based on clinical examination and general investigations such as ultrasound and CT Scan, instead of tumor pathology examination.Therefore it is necessary to confirm the results of anatomic pathology as gold standard in retinoblastoma cases for further management, determining the adjuvant therapy, prognostic factor, and determine the survival rate of retinoblastoma patients. 9,10atients with HRF must receive an adjuvant chemotherapy. 11his study purposeis to collect an overview of PRF and HRF in retinoblastoma patients in Sanglah General Hospital.

METHODS
This study is a retrospective descriptive study involving 20 cases of retinoblastoma that enucleated at Sanglah General Hospital and the laboratory results from Pathological Department Sanglah General Hospital.This study used consecutive sampling from 1 st January 2014 until 31 st December 2017.Qualified samples which fitted the inclusion criteria were collected.Incomplete data was excluded.

RESULT AND DISCUSSION
The study plan was approved from ethical committee of Udayana University/Sanglah General Hospital.There were 20 cases, retrieved from the previous medical record (2014-2017) and patient characteristics presented in Table 1.
The minimum age was seven months, while maximum age was 12 years (Age Range 7 months -12 years) while the mean age was 3.03 ± 2.69 years.The higher frequency of retinoblastoma cases founded inaged 2 -5 years old 13/20 (65.0%), while 4cases (20.0%) were found less than 2 years old.Most of the patients came from Bali (80.95%),only 3 patients (14.29%) were coming from West South East Nusa andone patient (4.76%) was coming from Timor Leste.

Sex and Family History
There were 10 female patients (50.0%) and 10 male patients (50.0%) suffering from Retinoblastoma involving in this study.The frequency on both sexes is almost the same with    male to female ratio was 1:1 (Table 1).There is no sex preference regarding retinoblastoma.From all patients, no one has family history of retinoblastoma.Laterality There were only 2 cases of bilateral retinoblastoma and 18 cases with unilateral neoplasms.The bilateral to unilateral neoplasm ratio was 1: 9.The age range of bilateral retinoblastoma was 1 -2 years while the range in unilateral tumor was 7 month -12 years old.In Unilateral cases, 6 patients (28.6%) were from right eye and 12 cases (57.1%) were from left eye.The percentage of bilateral retinoblastoma was 9.5%, and unilateral retinoblastoma was 90.5%.Extraocular invasion founded in 8 patients (35%)(Table 1).

Differentiation
There was cell differentiation suspected as active malignant cells in microscopic readings for 17 patients and only 3 patients with well-differentiated cell.On review of pathological slides, 3 eyes (4.3%) had well-differentiated tumor, 3eyes (4.3%) had Flexner-Wintersteiner rosettes differentiation which is related to retinoblastoma, 2 eyes (9.5%) had Homer-Wright rosettes differentiation, 12 eyes (61.9%) had undifferentiated tumor.

Pathological Risk Factor
Pathological risk factor of retinoblastoma present in all enucleated eyes, including 9 cases (45.0%) of anterior chamber invasion, vitreous body involvement in 6 eyes (28.6%), 12 cases (60.0%) of massive choroid infiltration, 12 cases (60.0%) of optic nerve invasion, 10 cases (50.0%) of scleral invasion, and extraocular extension in 7 cases (33.3%).Majority of the samples had more than one PRF at the same time, which are 2 PRF present in 1 eye (5.3%), 3 PRF present in 3 eyes with major choroid invasion, 46% with optic nerve invasion and 7% with sclera invasion and extra sclera extension. 12Kashyap et al. found only 3% has anterior segment invasion, and the remaining cases (97%) had more than one PRF. 15his study found high-risk PRF in 20 patients (100%), all of them have optic nerve invasion.However, the optic nerve invasion did not divide into pre and post lamina because not listed in the pathological report.Inferential analysis showed that major scleral invasion has significant association with tumor cell malignancy (p = 0.025).The result of current study is similar with study conducted by Suryawanshi et al. which found the incidence of trans-scleral invasion was 23.4%, higher than other studies which are about 15%. 14It can be caused by high stage retinoblastoma when enucleation performed.
Cuencha et al. divides the sclera invasion into two, namely the intra-sclera invasion if tumor cells are still confined within the sclera, not passing episclera, and trans-scleral invasion if tumor cells have penetrated the layers of the sclera and began to invade the periorbital tissue. 18Pathology reports did not describe in detail whether the invasion was found intra-sclera or trans-sclera.Invasion of the sclera especially trans-sclera is an important risk factor as predictors of recurrence, metastasis, and death, so it is necessary to give more intensive adjuvant chemotherapy to improve survival for patient with trans-sclera invasion. 13,14revious research also found many cases of extraocular relapse after enucleation in patients with scleral invasion as well as the pre and post lamina optic nerve invasion, so that both are approved as a high-risk PRF. 15 It suggests that the invasion of tumor cells in the post lamina optic nerve has the higher risk of metastases to the central nervous system through the subarachnoid space, it can even recur retinoblastoma in orbital.
Invasion of the choroid as predictors of extraocular relapse is still controversial.Font suggests the spread of tumor cells through the blood vessels of the choroid and ciliary can cause metastasis to the lungs, bones, and other tissues through the lymphatic system and blood-borne. 16helfaoui et al. found four cases of extraocular relapse in 20 patients with major choroid invasion. 17he incidence of choroid invasion on research by Suryawanshi et al. was 35.1%, higher than the research in Western countries, but research in developing countries showed that generally around 23% -32 %. 14  Suryawanshi et al. found no significant extra-ocular relapse in patients with major choroid invasion. 14he parameters of the pathology specimen examination play an important role to determine the stage of tumor growth and metastasis.It is useful in determining the prognosis and plan of adjuvant chemotherapy; it can also be used to evaluate the enucleation technique and examine whether the distance of the optic nerve resection far enough.Chantada, et al., (2004) found the identification of PRF is helpful in selecting patients who underwent adjuvant chemotherapy, so it can avoid not necessary adjuvant therapy that can cause complications such as myelosuppression, the risk of sepsis during chemotherapy, severe contractures of the orbital due to radiotherapy, and the risk of secondary malignancies due to chemotherapy and radiotherapy. 11umor cell differentiation varies among reports from various countries.Differentiation is divided into well-differentiated and poorly differentiatedt umors.Well-differentiated tumors give an image of rosettes and fleurettes.Filho et al. reported a high incidence of poorly differentiated tumors (80%) because of the age of the patient is older. 19Yousef et al. found 74% of patients with well-differentiated tumors at a median age of 26 months. 20Kashyap et al. also concluded that well-differentiated tumors are more common in younger patients. 21Suryawanshi et al. reveal that poorly differentiated tumor is a useful predictor for prognosis, but the results were not statistically significant because of the small number of patients, so it is necessary to conduct studies with a considerable number of patients. 14ll patients in recent study had adjuvant chemotherapy after enucleation, but the data recorded in the medical record was not completed makes it difficult to further follow up.Future research with long-term follow-up should be carried out to obtain useful information.

CONCLUSION
High-PRF of retinoblastoma present in all enucleated eyes (20 cases) in this study.There was cell differentiation suspected as active malignant cells in microscopic readings for 17 patients and only 3 patients with well-differentiated cell.More than one PRF were found in one sample at the same time.It revealed optic nerve and choroid invasion were the major PRF found in retinoblastoma, but, there is a significant association between scleral infiltration and tumor cell differentiation (p = 0.025).There was no statistical difference of PRF based on age, gender, and laterality.Further study with larger sample size is needed to investigate the PRF of retinoblastoma.

Fig. 4 .
Fig. 4. Low power of dense masses of small round cells tumor in poorly differentiated retinoblastoma

Table 1 .
Characteristic of Enucleated Retinoblastoma Patient

Table 2 .
PRF Percentage and Tumor Differentiation