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   Table of Contents      
BRIEF COMMUNICATION
Year : 2017  |  Volume : 65  |  Issue : 8  |  Page : 764-767

Molecular pathologic interpretation of new retinoblastoma rosettes


1 Department of Ocular Pathology, Uvea and Neuro-Ophthalmology Services, Sri Sankaradeva Nethralaya, Guwahati, Assam, India
2 Sri Sankaradeva Nethralaya, Guwahati, Assam, India

Date of Submission20-Apr-2016
Date of Acceptance27-Jun-2017
Date of Web Publication18-Aug-2017

Correspondence Address:
Dipankar Das
Department of Ocular Pathology, Uveitis and Neuro-Ophthalmology Services, Sri Sankaradeva Nethralaya, Beltola, Guwahati - 781 028, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijo.IJO_316_16

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  Abstract 


Newly described retinoblastoma (RB) rosettes have various kinds of pathological significance. Some of their characteristics have been revealed in this immunohistochemistry study. Five paraffin-embedded eyeballs with RB and new rosettes were studied for neuron-specific enolase (NSE), p53, p16, BAX, c-Myc, glial fibrillary acidic protein, synaptophysin, and chromogranin. They were compared and interpreted using control specimens. NSE, P53, and P16 were significantly expressed in the cells of the new rosettes. The presence of new RB rosettes that mostly have histopathological high-risk factors and p53 positivity may be a strong marker of poor prognosis of RB.

Keywords: Differentiation, immunohistochemistry, prognosis, rosette


How to cite this article:
Das D, Deka P, Bhattacharjee K, Barman MJ, Bhattacharjee H, Misra D, Saxena RK, Bhattacharyya A, Deka A. Molecular pathologic interpretation of new retinoblastoma rosettes. Indian J Ophthalmol 2017;65:764-7

How to cite this URL:
Das D, Deka P, Bhattacharjee K, Barman MJ, Bhattacharjee H, Misra D, Saxena RK, Bhattacharyya A, Deka A. Molecular pathologic interpretation of new retinoblastoma rosettes. Indian J Ophthalmol [serial online] 2017 [cited 2020 May 24];65:764-7. Available from: http://www.ijo.in/text.asp?2017/65/8/764/213252



Retinoblastoma (RB) constitutes 3% of all childhood cancers and has drawn the attention of both clinicians and basic scientists.[1],[2],[3],[4] The present success story of RB is much better than what was seen 100 years ago.[1],[2],[3],[4],[5] However, the scenario is a bit different in underdeveloped countries where even today, the disease is diagnosed at late stages and thereby affects the survival of children.[1],[2],[3],[5]

In 2014, we described and published the third true rosette [5] in RB, which had a characteristic morphology distinct from two other true rosettes which had been described over a century earlier. The new rosette described by Das et al. was further studied with molecular pathology interpretation under the Indian Council of Medical Research (ICMR) project conducted in two centers of India as a technology transfer assignment.

The aim is to study new RB rosettes by molecular pathology interpretation with immunohistochemistry (IHC).

Design

This was an institutional and laboratory-based molecular pathology study by the IHC in a technology transfer project under ICMR.


  Methods Top


Five paraffin-embedded tissues of Group E RB diagnosed clinically and pathologically with new rosettes, and histopathological high risk factors were selected and IHC was carried out by standard kit method.[6]

IHC for neuron-specific enolase (NSE), p53, p16, BAX, c-Myc, glial fibrillary acidic protein (GFAP), synaptophysin, and chromogranin were done, interpreted and documented. In the study, two cases of RB specimen with undifferentiated tumors with choroidal invasion and two cases of differentiated RB without new rosettes were taken as control.


  Results Top


All the cases in the cohort had Group E RB with new rosettes and histopathological high-risk factors. In all five samples, the relative size of the new rosettes was compared with Flexner–Wintersteiner (FW) and Homer Wright (HW) rosettes. It was found that the newer rosettes were comparatively larger than the FW and HW rosettes, as described earlier [Figure 1]. Immunomarkers which expressed strongly with new rosettes in the sections were positive for NSE, p53, and p16. P53 (immunostain: Anti-p53 protein, BioGenex, CA, USA), and a tumor suppressor gene was expressed in all RBs with new rosettes (mostly++). In the controlled specimen, undifferentiated tumors with choroidal invasion had significant p53 expression (+++). In the other controlled specimen group, the differentiated tumor without the new rosette did not express p53. The results of the specimen are shown in [Figure 2] and [Figure 3].
Figure 1: Relative size of new rosette described by author with Flexner–Wintersteiner rosette. New rosettes were larger than Flexner–Wintersteiner and Homer Wright rosettes (H and E, ×400)

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Figure 2: The immunohistochemistry of the new rosettes in 5 retinoblastoma cases with control (×400)

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Figure 3: Tabular form, all retinoblastoma cases with new rosettes indicating histopathological high-risk factors and immune reactive scoring for neuron-specific enolase, P-53, P16, BAX, c-Myc, glial fibrillary acidic protein, synaptophysin, and chromogranin with controls (please note: Immune reactive staining score: 0 with positive staining (0%–20%); + moderate staining [21%–50%]; ++ strong positive staining [51%–81%]; +++ very strong staining [81%–100%])

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  Discussion Top


Various IHC studies in RB pathology have shown prognostic clues for understanding the biology of this childhood cancer.[1],[2],[3],[4],[5],[6],[7],[8] We took up the study to know the characteristics of newly described rosettes with various known immunomarkers for RB. The new rosettes were associated with histopathological high-risk factors and morphologically different from FW and HW rosettes.[5] FW and HW rosettes were true rosettes described almost 100 years earlier.[1],[2],[3] Apart from true rosettes, there were fleurettes described in the late 60 s.[1],[2],[3] We had described the third true rosettes in RB in 2014.[5] FW and HW rosettes, along with fleurets, were seen in differentiated RB, but our described rosettes are in evolutionary processes which were seen in undifferentiated tumor with histopathological high-risk factors. FW rosettes had clear lumen surrounded by retinoblast cells whereas HW rosettes lacked clear lumens and had central neural fibrillar component. Newly described rosettes by Das et al. had retinoblast cells within the clear lumens and the cells within the hollowed space varied [Figure 4]. The newer rosettes were three to four times larger than conventional FW and HW rosettes. Fleurettes, on the other hand, were made up of eosinophilic processes in H and E stain in a bouquet-like arrangement, as if coming out of a fenestrated membrane. They were exclusively found in differentiated RB or the benign counterpart of RB called retinoma or retinocytoma.
Figure 4: Variation of central cells within clear lumen in new rosettes of retinoblastoma from single to multiple cells (H and E, ×400). They are in stages of evolution in dedifferentiation

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It was natural for us to query whether new rosettes were also correlated with molecular biologic changes seen with RB and other high-risk factors. We tried to see NSE, p53, p16, BAX, c-Myc, GFAP, synaptophysin, and chromogranin in our cohort and compared them with the controls: first, differentiated RB with FW and HR rosette, but without new rosette and second, undifferentiated RB with histopathological high-risk factors such as massive choroidal involvement.

NSE, p53, and p16 were expressed in RB with new rosettes, but p53 showed a very interesting and strong correlation with new rosettes RB and the control specimens. Undifferentiated control tumors with choroidal invasion had significant p53 expression (+++) which was consistent with other studies from India.[5] In another controlled specimen group, the differentiated tumor without the new rosette did not express p53.

P53 gene is a well-researched tumor suppressor gene positioned on the long arm of chromosome 17 and is known to be most marked for genetic modification in cancer genesis. Homozygous failure of p53 gene is seen in all kinds of cancer and also in RB.[9],[10],[11] Inactivating mutation effects in both p53 alleles are often acquired in somatic cells. In some situations, because of aggressiveness of the tumor, it may succeed to a mutant p53 allele.[10],[11] For RB, gene inheritance of one mutant allele predisposes the individual to build up a malignant tumor, as only one additional hit in the propagation of tumor is needed to deactivate the second normal allele.[10],[11] P53 acts as a doorkeeper against cancer. P53 abnormalities can lead to altered signal transduction pathways, loss of apoptosis, DNA repair, cell growth, cell proliferation and is often correlated with the advance of the tumor process.[10],[11]

P53 localizes at the nucleus, so all the tumors with the author's published rosettes which were in form of worse differentiation tended to express the molecule and contribute to the poor prognosis of RB.

Acknowledgment

The authors would like to thank Sri Kanchi Sankara Health and Educational Foundation, Guwahati, India; ICMR and Sankara Nethralaya, Chennai, India.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Biswas J, Das D, Krishnakumar S, Shanmugam MP. Histopathologic analysis of 232 eyes with retinoblastoma conducted in an Indian tertiary-care ophthalmic center. J Pediatr Ophthalmol Strabismus 2003;40:265-7.  Back to cited text no. 1
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2.
Eagle RC Jr. High-risk features and tumor differentiation in retinoblastoma: A retrospective histopathologic study. Arch Pathol Lab Med 2009;133:1203-9.  Back to cited text no. 2
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3.
Shields CL, Shields JA, Baez KA, Cater J, De Potter PV. Choroidal invasion of retinoblastoma: Metastatic potential and clinical risk factors. Br J Ophthalmol 1993;77:544-8.  Back to cited text no. 3
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4.
Gupta R, Vemuganti GK, Reddy VA, Honavar SG. Histopathologic risk factors in retinoblastoma in India. Arch Pathol Lab Med 2009;133:1210-4.  Back to cited text no. 4
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5.
Das D, Bhattacharjee K, Barthakur SS, Tahiliani PS, Deka P, Bhattacharjee H, et al. Anew rosette in retinoblastoma. Indian J Ophthalmol 2014;62:638-41.  Back to cited text no. 5
[PUBMED]  [Full text]  
6.
Elias JM. Immunohistochemistry: A practical approach to diagnosis. In: Principles and Techniques in Diagnostic Histopathology. 2nd ed. Chicago: ASCP Press; 2003. p. 14-33.  Back to cited text no. 6
    
7.
Seema R, Parul S, Nita K; Kamlesh. High-risk histomorphological features in retinoblastoma and their association with p53 expression: An Indian experience. Indian J Ophthalmol 2014;62:1069-71.  Back to cited text no. 7
[PUBMED]  [Full text]  
8.
Shields CL, Mashayekhi A, Au AK, Czyz C, Leahey A, Meadows AT, et al. The International Classification of Retinoblastoma predicts chemoreduction success. Ophthalmology 2006;113:2276-80.  Back to cited text no. 8
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9.
Eagle RC Jr. Retinoblastoma and simulating lesions. In: Eye Pathology-An Atlas and Text. 2nd ed. Philadelphia: Lippincott William and Wilkins; 2011. p. 207-33.  Back to cited text no. 9
    
10.
Yang X, Zhang Z, Zeng Q. A study of expression of p53, c-myc and PCNA in retinoblastoma. Zhonghua Yan Ke Za Zhi 1999;35:252-4, 14.  Back to cited text no. 10
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11.
Madhavan J, Ganesh A, Roy J, Biswas J, Kumaramanickavel G. The relationship between tumor cell differentiation and age at diagnosis in retinoblastoma. J Pediatr Ophthalmol Strabismus 2008;45:22-5.  Back to cited text no. 11
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

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