Vitreous fluid for primary intraocular lymphoma

Sara Kwong, MD, Cytopathology Fellow
Alaa Afify, MD, Professor and Director of Cytology

Introduction:  

Primary intraocular lymphomas (PIOLs) are a subset of primary central nervous system lymphoma arise from retina, vitreous, or optic nerve-with or without central nervous system (CNS) involvement.1 Most PIOLs is usually an aggressive diffuse large B-cell lymphoma although a small subset of the PIOLs can be T cell origin.2 The diagnosis of PIOL is often challenging because the clinical symptoms overlap with other pathologic conditions such as viral induced uveitis or autoimmune uveitis. Patients are initially treated with steroids or antiviral medication with no clinical improvement. The presence of malignant cells or tissue is required for the diagnosis of PIOLs. In addition, immunohistochemical stains, flow cytometric analysis, cytokine analysis, and immunoglobulin heavy chain gene rearrangements can provide invaluable information to support the diagnosis.3 Approximately 60-80% of PIOL will have CNS involvement and the accurate diagnosis is imperative for patients to receive timely treatment, which may lead to an improved mortality rate.4

Lab Best Practice:  

Specimen may be obtained via fine needle aspiration of the vitreous fluid or pars plana vitrectomy. The retrieved specimen must be delivered to laboratory in a timely manner to minimize cellular degeneration and to maximize the diagnostic yields.5 Even with the maximum effort, the specimen could have insufficient material to evaluate due to hypocellularity of the sample. Therefore, it may require several biopsies to reach an unequivocal diagnosis.6 It is important to note the prior treatment history, especially for patients who has received steroid because steroids have cytolytic effect on malignant lymphoma cells and could lower the diagnostic yield.7

Microscopically, PIOL show large lymphocytes (2-4 times the size of a normal lymphocyte) with scant cytoplasm, increased nuclear to cytoplasmic ratio, and coarse, immature chromatin with prominent nucleoli.8 These malignant cells can be identified using hematoxylin-eosin stain or Papanicolau stain, however, Giemsa or Diff-Quik stain is better to reveal the cytologic details of the cells.2 Cytology specimen has shown to have variable sensitivity in detecting intraocular malignancy that ranges from 31 to 66.7%,9 and one study reported a sensitivity of 83.3% of detecting PIOL.10 In addition, immunohistochemical stains can be performed, if there is a sufficient number of tumor cells, which often shows positivity immunohistochemical activity for B-cell markers, such as CD20 and CD22.

Flow cytometry is a very helpful technique to diagnose B-cell lymphomas and to discriminate infections and uveitis,11 however, it has shown limited utility in diagnosing PIOL due to the low cellularity nature of the specimen, which often yields limited or nondiagnostic findings.8

Cytokine analysis may also provide helpful information. B-cell lymphoma cells secretes high level of interleukin (IL)-10 and it is significantly increased in the vitreous fluid from patients with PIOL.12 On the contrary, in inflammatory conditions, such as uveitis, high level of IL-6 is produced. It has shown that the ratio of IL-10 to IL-6 greater than 1 is suggestive of malignancy. One study shows that IL-10 to IL-6 ratio of greater than 1 has sensitivity of 74.3% and a specificity of 75.0% in diagnosing PIOL.13

Polymerase chain reaction (PCR) is another powerful technique to detect the monoclonal gene rearrangement of immunoglobin heavy chain (IgH), and the presence of which is highly supportive of lymphoma cells. One study showed that 100% (50 out of 50 cases) tested PIOL cases had monoclonal IgH gene rearrangement.14 This test requires a minimum of 15 atypical lymphoid cells.15

Recently, the detection of a mutation in myeloid differentiation factor 88 (MYD88) gene (usually the canonical L265P mutation) has shown to provide diagnostic value. One study showed that this mutation was detected in 69% of primary vitreoretinal lymphoma, the most common form of intraocular lymphoma, with or without CNS involvement.16 This test utilizes the minimum amount of genomic DNA (10-20ng) for pyrosequencing.17

Conclusion:  

In summary, the diagnosis of PIOL can be extremely challenging, especially if the specimen yields low cellularity. Although a repeat sampling can be performed and may yield more cellular specimen for a definitive diagnosis, it is not always clinically feasible. On the other hand, several ancillary studies can be performed to improve the diagnostic specificity; however, these results must be interpreted in combination with clinical course, radiologic and cytologic evaluations.

References:  

  1. Swerdlow SH, Campo E, Harris NL, Jaffe ES, and PileriS A, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th Lyon, 2017.
  2. Faia LJ and Chan CC. Primary Intraocular Lymphoma. Arch Pathol Lab Med. 2009;133(8):1228-1232.
  3. Sen HN, Bodaghi B, Hoang PL, and Nussenblatt Primary intraocular lymphoma: diagnosis and differential diagnosis. Ocul Immunol Inflamm. 2009;17(3):133-141. PMID: 19585354.
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  12. Cassoux N, Giron A, Bodaghi B, Tran THC, Baudet S, et al. IL-10 measurement in aqueous humor for screening patients with suspicion of primary intraocular lymphoma. Invest Ophthalmol Vis Sci. 2007;48(7):3253-3259.
  13. Wolf LA, Reed GF, Buggage RR, Nussenblatt RB, and Chan CC. Vitreous cytokine levels. Ophthalmology. 2003;110(8):1671-1672.
  14. Chan CC. Molecular pathology of primary intraocular lymphoma. Trans Am Ophthalmol2003;101:275-292.
  15. Wang Y, Shen D, Wang VM, Sen HN, Chan CC. Molecular biomarkers for the diagnosis of primary vitreoretinal lymphoma. Int J Mol Sci. 2011;12(9):5684-5697.
  16. Fend F, Ferreri AJ and Coupland SE. How we diagnose and treat vitreoretinal lymphoma. Br J Haematol. 2016;173(5):680-692.
  17. Cummings PJ, Ahmed R, Durocher JA, Jessen A, Vardi T, et al. Pyrosequencing for microbial identification and characterization. J Vis Exp. 2013;(78).
By | 2020-04-22T07:33:02+00:00 May 15, 2020|0 Comments

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