Despite its status as the most widely accepted biomarker in the workup of gliomas, 1p/19q codeletion has never been shown to be a truly predictive marker in the sense that HER2 is, wherein amplification predicts a specific response to trastuzumab in breast and upper gastrointestinal cancers. Thus, there has not been a broad-based initiative to standardize 1p/19q testing, which has resulted in remarkable interinstitutional heterogeneity of assays, probes, and cutoff criteria. For example, even when using FISH (specifically the commercially-available 1p36/1q25 and 19q13/19p13 probes from Vysis, Abbott Park, IL), many laboratories focus on 1p/1q and 19q/19p ratios in their evaluation whereas others rely solely on the percent of tumor nuclei showing relative 1p and 19q loss, with a cutoff generally around 40% (35
). Our original ratio cutoffs were established as 3 standard deviations beyond the ratios normally seen in non-neoplastic control tissues. To enhance the rigor of the assay, 20 non-neoplastic control samples were used. Applying hindsight, it is clear that increasing the number had the inevitable effect of decreasing standard deviations, i.e. the cutoffs were too inclusive, producing lower concordance with PCR-based LOH data and less prognostic power. Lowering the ratio cutoffs improved concordance with LOH and the prognostic power of FISH ().
From a biological perspective a more stringent pair of ratio cutoffs makes sense, because the unbalanced translocation that produces 1p/19q codeletion is believed to occur at a relatively early stage of gliomagenesis and should therefore be present in most if not all glioma cells. Thus, “true” 1p36/1q25 and 19q13/19p13 ratios ought to be closer to the hypothetical “pure” ratio of 0.5. In contrast, random interstitial deletions could occur at any point during tumor development and would thus be expected to show more cell-to-cell variation, with some cells in a tumor containing partial deletions but not others. It is therefore not surprising that 1p36/1q25 and 19q13/19p13 ratios tended to be higher in gliomas that had apparent codeletion by FISH but not by LOH vs. tumors with codeletion by both modalities (Supplemental Fig. 2
There are no perfect criteria or cutoffs that produce 100% sensitivity and specificity for 1p/19q FISH (as in any diagnostic test). For example, applying the modified FISH criteria produced a better p value in survival stratification of grade III oligodendrogliomas because it avoided calling 2 cases as codeleted that were intact by LOH analysis; those patients died 2 months and 1.3 years after diagnosis. Yet it also excluded a case that was actually codeleted by original FISH criteria and by LOH analysis; that patient survived 5.3 years. Eschewing ratios altogether, instead using cutoffs of at least 40% tumor cells with relative 1p and 19q deletion, produced a comparable degree of prognostic stratification to the 0.75 ratio cutoffs in anaplastic oligodendrogliomas (p = 0.009 for % deleted vs. 0.007 for ratios, data not shown). Furthermore, both the modified ratios and ≥40% relative deletion criteria had 95% concordance with each other in all oligodendrogliomas. Thus, it probably does not matter whether one employs ratios or % deletion because they produce essentially the same overall results. What seems more important is that these cutoffs have been determined in this study using prognostic stratification and whole-arm assays as the primary litmus tests rather than relying on non-neoplastic tissues.
Although in most cases there was good concordance between FISH and PCR-based LOH, in this cohort 1p/19q PCR-based LOH showed stronger prognostic value in grade III anaplastic oligodendrogliomas. That neither test was able to significantly stratify grade II oligodendrogliomas () is not surprising because 1p/19q codeletion appears to have less prognostic impact in low-grade gliomas and may only show significance with prolonged follow-up intervals (8
). Indeed, an analysis of our grade II subset suggested insufficient power (0.67) to stratify by 1p/19q status given our cohort size, median follow-up interval and the relatively longer survival in this subgroup. On the other hand, because grade III tumors have shorter survival overall and a much sharper survival difference by 1p/19q (or 10q) status, the subset of 32 cases was adequately powered (0.91), Still, testing for codeletion at least has diagnostic value, particularly in gliomas in which morphologic features are equivocal between astrocytoma and oligodendroglioma (19
Oligodendrogliomas with true 1p/19q codeletion but 10q LOH are rare to begin with and sometimes have fewer classic features of oligodendrogliomas, suggesting that the diagnosis of oligodendroglioma in the face of 10q deletion may be reconsidered (7
). Our present results support and extend these findings, specifically making the critical point that most cases called “codeleted” by FISH are false-positives if 10q is also deleted. Whereas rare gliomas do indeed have concomitant whole-arm 1p/19q codeletion and 10q LOH, many so-called 1p/19q-codeleted gliomas are likely not truly whole-arm codeleted when 10q is also lost (; ).
Prior work showed that increased EGFR expression is paradoxically correlated with improved survival in anaplastic oligodendrogliomas (33
). The current data reconfirm this on multivariate analysis (). The reasons for this are not clear but it is not due to nonrandom segregation of 1p/19q LOH (p = 0.46), 10q LOH (p = 0.54), or younger patient age (p = 0.41).
Although PCR-based LOH is more powerful than FISH, and assays such as single nucleotide polymorphism, array comparative genomic hybridization, and multiplex ligation-dependent probe amplification show great promise (44
), our results indicate that it is likely that FISH will remain a widely-used technique in the workup of gliomas. Standardized protocols for 1p/19q FISH have recently been recommended (36
), which should be of great assistance to laboratories worldwide; however, universal standardization of 1p/19q FISH probe loci, interpretation criteria and reporting criteria are also needed. Based on our data, we suggest the following scoring and reporting parameters for 1p/19q FISH: 1) If using 1p36/1q25 and 19q13/19p13 ratios, they should each be <0.75. 2) If using relative deletion, both 1p36 and 19q13 should be deleted in at least 40% of cells. 3)10q interrogation, either by PCR analysis or FISH (e.g. testing for PTEN deletion) should be done in cases where 1p/19q FISH is positive or equivocal for codeletion, and if 10q is deleted, the 1p/19q data should be interpreted with great caution; Indeed, it may even be advisable to test 10q status in oligodendrogliomas regardless of the 1p/19q results, as it may be an independent adverse prognostic factor (, ).
These are useful parameters to serve as a starting point for the development of universal scoring and reporting criteria for 1p/19q FISH. Once such standards are in place, they will enhance the quality of prognostic information provided to clinicians and patients. They will also improve inter-institutional reproducibility of glioma research, especially work that focuses on 1p/19q codeletion, 10q deletion, and their roles in glioma biology.