Biopsy techniques and yields in diagnosing primary intraocular lymphoma
A review of current biopsy techniques that are used in obtaining specimens from which to make a diagnosis of primary intraocular lymphoma (PIOL) is presented. Methods for obtaining and subsequently testing vitrectomy specimens are discussed. In addition, the yields of external and internal approaches for obtaining chorioretinal tissue, and diagnostic vitrectomies, are reviewed.
KeywordsPrimary intraocular lymphomaVitrectomyChorioretinal biopsyTransvitreal retinal biopsy
primary central nervous system lymphoma
primary intraocular lymphoma
polymerase chain reaction amplification.
retinal pigment epithelium
Primary intraocular lymphoma (PIOL) is a subset of primary central nervous system lymphoma (PCNSL) with predilection for intraocular regions that sit behind the blood-retina barrier [1–4]. PIOL is a unique malignant lymphoproliferation because of its affectation of an immune privileged site, involving the subretinal pigment epithelium (RPE), retina, vitreous, and optic nerve. Frequently masquerading as a uveitis, PIOL is often misdiagnosed for intraocular inflammation and is treated with corticosteroids  or, infrequently, PIOL can masquerade as a viral retinitis and is treated with antiviral medication . It is not until the presumed uveitis fails to respond to corticosteroid therapy that another cause is sought. Prompt diagnosis of PIOL is imperative because consultation with a neurooncologist and initiation of chemotherapy and/or radiation therapy can extend the patient’s life. Furthermore, most cases of PIOL will eventually involve the brain (PCNSL) [1, 7], which has a poor prognosis. Thus, if disease can be halted within the eye, there is a possibility that morbidity and mortality may be improved in patients with PIOL. Diagnosis of PIOL requires histopathologic evidence of the malignant lymphoma cells. A tissue biopsy must be obtained from which to make a pathologic diagnosis and perform further testing including molecular analyses. When undertaking a biopsy it is important to have a diagnostic plan laid out. Because an ocular pathologist will ultimately receive the biopsy specimen and will be making the diagnosis in a suspected case of PIOL, it is important to involve this member of the patient’s care team prior to bringing the patient to the operating room. The ocular pathologist can help determine which affected intraocular structure may provide the best opportunity to result in the highest yield of lymphoma cells.
In this article, we review the biopsy techniques and yields that may be employed in order to provide tissue from which to make a diagnosis of PIOL in suspect cases. It is important to realize that, if a patient is suspected of having PIOL, a lumbar puncture with cytologic analysis of the cerebrospinal fluid (CSF) should be performed because of the high possibility of brain involvement in PIOL [1, 3, 7–9]. In addition, patients should also receive a brain magnetic resonance imaging (MRI) scan to determine whether lesions are present in the absence of obvious lymphoma cells in the CSF. When brain involvement has been ruled out by the above procedures, we focus on the involved eye from which to make a diagnosis.
Prior to the advent of pars plana vitrectomy in the 1970s pioneered by Machemer [10–15], enucleation [16, 17] was the first surgical procedure employed by ophthalmic surgeons to make a diagnosis of the so-called reticulum cell sarcoma (as PIOL was known prior to the 1970s) [8, 17, 18]. Enucleation could be performed in patients’ eyes in which there was no possibility of restoring vision (vision worse than count fingers) due to massive involvement or in eyes that had become intractably painful. In addition, in patients succumbing to their disease, enucleation could be performed to confirm a clinical suspicion of reticulum cell sarcoma. Enucleation, however, is not an ideal diagnostic procedure to undertake when a patient still has potential functional vision and an otherwise normal eye.
The vitreous remains the preferred tissue to sample when an eye exhibits chronic uveitis of unknown cause or intraocular malignancy or infection is suspected. Vitrectomy is also indicated when there is potential for treatment to be initiated or changed upon the diagnostic procedures used on the vitrectomy specimen. Indeed, often masquerading as a vitritis, PIOL cells are commonly found in the vitreous and this structure is most frequently biopsied to make a pathologic diagnosis of PIOL.
Cytologic examination of vitreous biopsy specimens has been employed to make a diagnosis of PIOL since the mid-1970s in cases that were initially diagnosed as uveitis [19, 20]. However, lack of improvement with typical corticosteroid treatment prompted vitreous biopsy with cytology, revealing the atypical lymphocytes associated with PIOL [19, 20]. Importantly, a final diagnosis of PIOL allows the appropriate treatment to commence (in the mid-1970s this was primarily radiation treatment [19, 20]). Vitrectomy can also yield a diagnosis of PIOL when lumbar puncture and cytologic analysis of CSF fail to reveal PCNSL cells .
Vitreous specimens may not always contain neoplastic cells and, thus, be negative for the diagnosis of PIOL. This might especially be the case if there is minimal vitreal involvement by the PIOL cells  or the cells have degenerated. Sometimes the quality of the cytology is poor, therefore making it unfeasible to make the diagnosis. In such events, it may be necessary to perform another vitrectomy and send to a well-qualified cytological laboratory .
Davis and colleagues submitted vitrectomy specimens from 27 patients suspected of having an intraocular malignancy to analysis by cytology . A final diagnosis of lymphoma was achieved in 13 patients with cytology yielding four true positive lymphoma cases (a sensitivity of 31%) and no false positive cases. The positive predictive value (PPV) and negative predictive value (NPV) of cytologic evaluation, then, was 100 and 60.9%, respectively. Thus, while a positive cytologic evaluation was certain for lymphoma, a negative evaluation certainly does not completely rule out the possibility of intraocular lymphoma. Earlier, in a series of 87 patients who, by clinical examination, were suspicious for PIOL it was found by cytologic analysis of the vitreous specimen that 42 were positive for PIOL, another three were suspicious for PIOL, and 42 were negative .
A negative cytology, then, is not always reassuring. When suspicion for PIOL still runs high, it is important to consider other adjunctive testing to which the vitrectomy specimen might be submitted. In addition, while the ability to acquire vitreous specimens exists at many surgical ophthalmology centers, the appropriate analyses and experienced pathologists to evaluate such specimens reliably may not. In these instances, it is important to consider sending the vitreous specimen to a center that has the capacity to evaluate it. However, even though the vitreous specimen may be sent to a major hospital with an experienced histopathologist, the very real possibility that any lymphoma cells within the vitreous biopsy could degenerate exists. It is imperative that the appropriate handing of the vitreous specimen occur and to be aware of adjunctive tests that may be performed at a nearby laboratory by those experienced with the basic techniques of molecular biology .
Diagnostic testing of vitrous biopsy specimen (sample)
B-cell malignancies can secrete high levels of IL-10 , an immunosupressive cytokine, while inflammatory conditions (such as uveitis ) are associated with high levels of IL-6 [39–41], a pro-inflammatory cytokine [42, 43]. We [24, 26, 44, 45] and others [27, 46] have shown that PIOL can exhibit high IL-10 levels with IL-10:IL-6 ratios greater than 1.0 being suggestive of PIOL . Cytokine levels and IL-10:IL-6 ratios are by no means diagnostic of PIOL, but they can be useful adjunctive tests in corroborating suspicion of PIOL and determining whether there is a durable response to treatment. For example, one of our patients had IL-10 levels that seemed to correlate with the amount of cells in the vitreous and degree of vision . The patient received systemic and intrathecal methotrexate (receiving six cycles). Over six years, however, vitreous biopsy showed that there continued to be intraocular disease. When IL-10 levels were high and IL-10:IL-6 ratios were much greater than 1.0, there were noted to be many more vitreous cells and relatively poor vision. After intravitreal methotrexate administration, the patient would respond by having low to undetectable levels of IL-10 and IL-10:IL-6 ratios less than 1.0, correlating with a relative lack of vitreous cells and improvement in vision. This patient, however, developed new cerebral lesions (PCNSL) and eventually expired. We have shown that cytokine levels can be significantly different between intraocular lymphoma and uveitis . By determining a IL-10:IL-6 ratio greater than 1.0 in suspected cases of PIOL, we correctly classified such cases as PIOL 74.7% of the time (with a sensitivity and specificity for the cutoff of 74.3 and 75.0%, respectively) . Figure 1 shows an appropriate step in which to submit part of the vitreous specimen to cytokine analysis.
It remains to be seen, however, what the PPV and NPV of PCR testing might be. Currently, cytologic evaluation is still the gold standard by which diagnoses of PIOL are made. If PCR, in fact, yields sensitivity far superior to that of cytology (while maintaining the same specificity, i.e., 100%) perhaps PCR analysis of vitrectomy specimens may supplant cytology for making a diagnosis of PIOL. Vitreous samples that contain very few lymphoma cells that are outnumbered by inflammatory cells may result in the polyclonal inflammatory cells overshadowing the monoclonality of the PIOL cells. Histo- and cytopathologic evaluations will never truly be supplanted because identification of malignant cells to describe immunohistological subtyping and to identify atypical cells for microdissection (which can improve the sensitivity of PCR analysis) will always be necessary. Indeed, at the NEI we are familiar with cytologic evaluation being noncontributory in making a diagnosis of PIOL. While we frequently employ molecular pathologic analysis (microdissection and PCR) to determine monoclonal rearrangement of the IgH gene [24, 25, 29] this test is still considered to be adjunctive to cytology. Appropriate time points to perform PCR are shown in Fig. 1.
External chorioretinal biopsy
While vitrectomy with the aforementioned evaluations may provide the ophthalmic surgeon with a diagnosis, this technique requires that there be malignant cells within the vitreous. However, failure to identify malignant cells in the vitreous can occur and may be due to degeneration of the malignant lymphoma cells, paucity of cells in the vitreous, or lack of involvement altogether of the vitreous. Lymphomatous involvement may be confined solely to the sub-RPE and chorioretinal biopsy (pioneered by Peyman and colleagues [52–55]) may yield tissue with which to make a diagnosis of PIOL. Interestingly, Peyman and colleagues were the first to report making a diagnosis of PIOL from tissue obtained via external chorioretinal biopsy .
The surgical technique for performing an external chorioretinal biopsy is fairly straightforward and is discussed with references to its essential points [56, 59]. First, provided that the fundus is clearly visible, laser photocoagulation is applied 1–3 days before surgery in a zone of the area to be biopsied. If the vitreous is too hazy, endolaser is placed immediately following vitrectomy. After a 360° conjunctival incision and isolation and tying of the rectus muscles a three-port pars plana vitrectomy is performed (in addition to endolaser application in the area to be biopsied if it was not placed prior to the surgery). The vitreous specimen (dilute and undilute) is sent for cytologic and microbiologic analyses as well as cytokine (IL-10 and IL-6) levels [1, 2, 24, 25, 30, 31, 57]. A nearly full-thickness scleral flap is made, leaving one side attached to act as a hinge. When the flap of sclera is retracted, the surgeon is able to visualize the choroids, which is practically bare. Next, a penetrating diathermy is placed through the choroid and retina along the outer margin of the inner choroidal bed. Two incisions parallel to the limbus are made. Next, by inserting one blade of a 0.12 forceps through the incision, the full thickness of the choroid and retina may be grasped at one edge. Then, two more incisions, perpendicular to the limbus, are made with Vannas scissors, thereby yielding a block of chorioretinal tissue. Extreme care should be taken to grasp the full-thickness tissue only once with the forceps so that the architecture of the tissue remains intact [56, 59]. The scleral flap is then closed over the wound and is sutured closed followed by fluid-gas exchange.
Transvitreal retinochoroidal biopsy
Cassoux and colleagues have also used endoretinal biopsy to diagnose PIOL when vitrectomy was nondiagnostic . The patient had undergone vitrectomy twice that failed to reveal any malignant lymphocytes, but a high IL-10 level was detected . Cassoux et al. noted that their technique for performing an endoretinal biopsy involved the induction of a localized retinal detachment (if one was not already present) by injecting sodium hyaluronate into the subretina. The resultant bulging retina was subsequently excised by cutting around the perimeter with scissors and extricating the biopsy with forceps. Endolaser was then applied around the biopsy site .
Diagnostic testing of chorioretinal and endoretinal biopsy tissue
The biopsy tissue is immediately processed by an ophthalmic pathologist in the operating room. It is generally divided into three portions . One third of the tissue is fixated for routine histopathologic studies, including light and electron microscopic examinations. The second portion is snap frozen in optimal cutting temperature (OCT) embedding compound and is used for immunopathologic and molecular characterization. The third portion is sent for culture with the preference for viral and other microorganisms cultures and/or tissue culture .
Again, external chorioretinal and transvitreal retinochoroidal biopsies are typically performed when diagnostic vitrectomy in inconclusive but there remains a very high suspicion for PIOL. Risks of biopsying, which include hypotony, hemorrhages, endophthalmitis, retinal detachment, and cataract formation should be outweighed by the risk of allowing lesions that threaten the macula, or are infectious or malignant in etiology to progress. In addition, a plan should be designed so that proper testing of the biopsy tissue is carried out. This requires coordinating care amongst the surgeons, pathologist, microbiologist, and molecular biologists. The information obtained from the biopsy can often lead to a change in clinical treatment (such as initiating chemotherapy) with benefits not only seen in visual acuity improvement and tumor regression, but in potentially extending the life of the patient .
Endoretinal biopsy tissues are often small and delicate and require handling with extra care . The biopsy specimen may not be able to be divided into three portions, but, rather, only enough to be processed for frozen sections. These sections can then undergo routine histology, immunohistochemistry and molecular analysis.
In summary, vitreal, chroioretinal and/or endoretinal biopsy is needed to provide the diagnosis of PIOL if lymphoma cells are not found in the CSF. Prior to the biopsy procedure, it is critical to have a thorough discussion among the surgeons (vitreoretinal surgeon and uveitis specialist), pathologists and molecular biologists. The discussion and plan will help to minimize surgical risks and to carry out the biopsy tissue properly. The information obtained from the biopsy can often lead to a correct diagnosis, appropriate clinical treatment, and prolonging the PIOL patient’s life .
Support: The NEI intramural research program