METHODS

This study was approved by the institutional ethics committee of the Hospital do Câncer AC Camargo, São Paulo, Brazil. In order to be certain that any disease dissemination (distant metastasis or orbital disease) in the study patients originated in the enucleated eye, we studied only patients with unilateral intraocular retinoblastoma, treated solely by enucleation. We carried out a retrospective analysis of the medical records of all patients with unilateral retinoblastoma seen at the Ocular Oncology Sector from the Hospital do Cancer AC Camargo, São Paulo, Brazil, from January 1992 to December 1995. Fifty eight patients were identified. Of these, 34 were excluded for the reasons listed in Table 1.

Twenty four patients met the inclusion criteria and had sufficient data to be included in the study. These 24 patients were divided in two groups. Group I consisted of five patients who developed either local orbital disease or metastatic retinoblastoma at a mean of 10.4 months following enucleation (median 10; range 6–13). Group II consisted of the remaining 19 patients who did not develop disseminated disease in a mean follow up of 54 months. The patients are described in Tables 2 and 3.

All patients in the study were classified as Reese-Ellsworth group Vb (with the exception of two patients classified as Va, one in each group) at diagnosis and treated by enucleation alone.³⁵ No post-enucleation treatment was given in any case unless disease dissemination was documented.

One haematoxylin and eosin section of the enucleated eyes was examined for optic nerve and choroidal invasion risk factors using the classification proposed by Khelfaoui et al.¹⁴ When the maximal tumour height was not in the same plane as the pupil-optic nerve section, additional sections including the optic nerve were studied (Table 4).

In order to assess TAC, one additional tissue section, subsequent to the one for haematoxylin and eosin, was submitted for immunohistochemistry by the streptavidin-biotin-peroxidase complex method (Dako, Dane). The primary antibody used to highlight endothelial cells was the monoclonal antibody to CD 34 class II (Dako, Dane), at 1:50 dilution. Antigen retrieval was achieved by microwave treating the slides in a 0.01 mM citrate buffer (pH 6.0) at 1300 W for 9 minutes twice. Endogenous peroxidase activity was blocked by incubation of the slides in 0.3% hydrogen peroxide (H₂O₂) four times, 5 minutes each time. Normal goat serum was used to block non-specific antibody binding. Diaminobenzidine (DAB) 0.06% was used as the chromogen, staining microvessels dark brown. Finally, sections were counterstained with Harris’s haematoxylin. Tissue sections of a normal liver were used as the positive control. Negative control slides were generated by substituting phosphate buffered saline (PBS) solution for the primary antibody.

The CD 34 stained slides were scanned under ×100 magnification to identify the brownest vascular staining areas or “angiogenic hot spots.” The magnification was then increased to ×320. With the use of a 100 point graticule (KPL 16X Karl Zeiss, Germany), the intersections of the brown stained points with the graticule were counted in four fields from the same angiogenic hot spot resulting in a total of 400 points counted. A single investigator (VA), who was unaware of the clinical outcome, performed the histopathological review and measurement of the angiogenic capacity through the quantification of the tumour’s relative vascular area (TRVA).

The TRVA was calculated using the Fallowfield and Cook equation adopted from the previously described method of Chalkley^26,36 and used as a measurement of TAC:

Number of intersections of brown pigment with the graticule/number of counted points × 100%

Statistical analysis

The statistical analysis was performed using the NCSS 2000 program (NCSS Statistical Software, UT, USA). Mann-Whitney-Wilcoxon U test and Kolmogorov-Smirnov test were used for two group comparative analysis. The Cox proportional hazard regression model was used to access which variables were independent predictors of metastatic and/or orbital disease using an assumed level of significance of 0.05. The receiver operating characteristic (ROC) curve analysis was used to evaluate sensitivity and specificity of the TRVA as a predictor test for disease dissemination.

RESULTS

In the two groups of patients, the only variable that achieved statistical significance as a predictor of subsequent disease dissemination was the TRVA (Table 5). The mean TRVA was 4.5% in group I (median 4; range 4–6%) and 2.75% in group II (median 2.5; range 1.25–4.75%).

There was no difference between the two groups with regard to age at diagnosis, the greatest tumour diameter, choroidal invasion, or optic nerve invasion (Table 5).

By Cox proportional hazard regression, the only variable that independently predicted the development of orbital and/or metastatic disease was the TRVA (p = 0.008). Neither tumour diameter (p = 0.84), choroidal invasion (p = 0.77), nor optic nerve invasion (p = 0.62) achieved statistical significance.

ROC curve analysis was done to access the sensitivity and specificity of this finding. A TRVA of 3.9% was found to be the point of best balance between sensitivity (100%) and specificity (79%) for the risk of developing metastatic and/or orbital disease following enucleation (Fig 1). The area under the curve for the TRVA was 0.91.

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Figure 1

ROC curve for the tumour’s relative vascular area as a predictor of disease dissemination.

Metastatic disease was diagnosed in all five group I patients at mean of 10.4 months, with the maximum time of 13 months following enucleation (Table 2). The shortest follow up for any of the 19 group II patients, who did not develop metastatic and/or orbital disease was 16 months (Table 3). Thus we considered that an adequate follow up was obtained and that none of the tumour free patients in group II was tumour free because they had not been followed for a sufficient period of time. The location of relapsed extraocular disease and final outcome of each group I patients are listed in Table 2. Since only patient 3 in group I and patient 18 in group II were classified as Reese-Ellsworth Va, with the remaining classified as Vb, no conclusions could be drawn with regard to this variable.

DISCUSSION

After enucleation has been performed in patients with retinoblastoma, the ocular pathologist is called on to estimate the risk for metastatic disease to the clinician. Currently, the decision about the use of prophylactic therapy is based on the histopathological presence and extent of tumour cell invasion into the optic nerve and/or choroids.^{1–15,37–39} Most commonly only patients with optic nerve post-laminar invasion are recommended for prophylactic or adjuvant treatment.³⁷

Retrospectively, we can hypothetically evaluate the precision of optic nerve invasion, choroidal invasion, and the TRVA as criteria to define which patients would need prophylactic treatment in our series. If post-laminar invasion of the optic nerve had been the criterion used in deciding when adjuvant therapy was indicated in this series, three of the five patients who developed disease dissemination (numbers 2, 3, 4 of group I, Table 2) would not have been treated. Patient 19 (group II, Table 3), who did not develop disease dissemination, would have been unnecessarily treated. If massive choroidal invasion alone had been the treatment criterion, two patients in group I (2, 4, Table 2) would not have been treated and unnecessary treatment would have been given to four patients (5, 9, 13, 19 in group II, Table 3). If a TRVA equal to or greater than 3.9% had been used, all patients who did develop disease dissemination would have received adjuvant therapy shortly after enucleation and disease dissemination probably would have been avoided. Using the same criterion, four patients who did not develop disease spread without treatment would have received adjuvant therapy unnecessarily (patients 16–19 in group II, Table 3).

One possible limitation of our study is that only eight of our patients had post-laminar invasion of the optic nerve and/or massive choroidal invasion. This is probably the experience of most centres that treat this rare disease. The low incidence of disease dissemination after enucleation for intraocular retinoblastoma, and the great heterogeneity in its management, can further obscure the importance of optic nerve and choroidal invasion, since some forms of pre-enucleation and post-enucleation treatment can act as protective factors against disease dissemination.^12,37 In this series we did not include patients with optic nerve invasion up to the surgical margin nor did we include those with known extrascleral invasion.

Reports of metastatic and/or orbital disease after enucleation in the absence of any current histopathological risk factors do exist, exemplifying their lack of sensitivity. In Kelfaoui’s series, orbital or metastatic disease was detected in 23 of 172 patients, three of them had only minimal choroidal invasion and prelaminar optic nerve invasion.¹⁴ Shields et al reported two cases of metastatic disease in the absence of optic nerve or choroidal involvement of any kind.¹² MacKay et al also reported two patients with metastatic disease but no evidence of optic nerve and choroidal invasion. These authors assume that an inadequate histological sample was probably the cause.⁶ In our series, two of the five patients in group I had no optic nerve or choroid invasion and developed orbital disease (Table 2).

Inadequate histological sampling seems to be a major error factor when evaluating prognostic factors. Sampling all the specimens would solve this problem, but it is not practical nor is it adequate for routine use. This is why we chose a test that even when performed in a single tumour slide section can give reliable prognostic information, making it suitable for routine use.

Angiogenesis is a term that defines any new vessel formation from the pre-existing vascular bed.⁴⁰ Under physiological conditions it is usually limited to development, reproductive and cicatrisation periods.¹⁷ Tight control of angiogenesis is lost in some disease processes like rheumatoid arthritis, many retinopathies and cancer.²²

Two phases, avascular and vascular, divide the natural history of cancer.^19,21 In the avascular phase, tumour growth is greatly limited owing to lack of nutrients and the cells clones are incapable of metastasising. In the vascular phase, there is a shift to an angiogenic phenotype that results in exponential tumour growth.^{17–20,22,41}

There is published evidence of angiogenic capacity as a diagnostic marker in retinoblastoma.^29–31 The only study that investigated any correlation between the vascular component and prognosis in retinoblastoma failed to prove it, perhaps because of the choice of an antibody that was not specific for endothelial cells and the use of non-quantitative methodology.⁹

It is accepted that cancer cells exist in a hypermutable state. This can lead to the accumulation of genetic alterations. If any of these random mutations result in the creation of tumour clones with an increased growth rate and the ability to metastasise, these clones will rapidly become the dominant cell in the tumour.³⁹

The TAC has been shown to be, at least in part, dependent on some of those genetic alterations, such as inhibition of the p53 tumour suppressor gene.^42–44 Thus, the complex metastatic process which involve shedding of cells, escaping the host immune system, invasion through vessels walls by the neoplastic cells, extracellular matrix degradation, and recruitment of a vascular supply seems much easier for a highly angiogenic cell lineage.^18,19,40

The use of the TRVA (assessed by Chalkley count) as a measurement of the TAC has been shown to be sensitive, reproducible, and objective.^25,26,45 This morphometric method to estimate the volume of a three dimensional structure from a thin two dimensional slice was adapted from the work of geologists and metallurgists.^36,46 It is based on the Delesse principle: “the planimetric fraction of a section occupied by sections of a given component corresponds to the fraction of the tissue volume occupied by this component.”⁴⁶

According to the Cox proportional hazards regression analysis, only the TRVA proved to be an independent prognostic predictor for disease dissemination (p = 0.008). Although all patients who developed disseminated disease had a TRVA equal or greater than 4%, four patients in group II who did not subsequently develop disease spread had measurements equal to or above this value (Table 3). Of particular note is patient 19 who had a TRVA of 4.75%, post-laminar optic nerve invasion, massive choroidal invasion, and did not develop metastatic disease. Another interesting issue is that patients 2, 3, and 4 in group I developed their first manifestation of extraocular disease in the orbit. Although this could represent a non-detected microscopic direct extraocular extension present at the time of surgery rather than metastatic dissemination, the TRVA would still be capable of predicting a poor prognosis in these patients. Thus, it is clear that whatever the dissemination mechanism an increased ability to recruit vessels is crucial for tumour growth.

As is the case in many human cancers, probably the most important prognostic predictor in retinoblastoma is still an early diagnosis.^10,47–49 Our series contains a higher proportion of patients who developed disseminated disease after enucleation than some similar series.^11,12,14 This fact may reflect more advanced intraocular disease in our patients since the average age at diagnosis is greater than the mean for unilateral tumours in other series.^37,48 It is logical to assume that since a large tumour has undergone more cellular divisions, its probability of accumulating the genetic alterations required for metastatic spread is increased.

The TRVA seems to be a promising prognostic marker that should be evaluated in a larger sample of retinoblastoma eyes. It holds the promise of improving the accuracy of our prediction in detecting those patients who truly need adjuvant chemotherapy to protect against micrometastatic disease.