The diagnostic value of 11q13 amplification and protein expression in the detection of nodal metastasis from oral squamous cell carcinoma: a systematic review and meta-analysis

New diagnostic biomarkers to improve the diagnosis of nodal metastasis in patients with OSCC are pivotal for a better and more individualized treatment of the neck [3]. Amplification of 11q13 is common in head and neck cancer, and several studies showed a correlation with metastasis and poor survival. However, results vary between studies, and no coherent review has been performed at present with regard to the diagnostic value of 11q13 amplification, amplification of individual genes located on 11q13, or overexpression of its genes in the detection of nodal metastasis from oral cancer. Little is known about the NPV of these alterations, which is the most important diagnostic value to safely omit an elective treatment of the neck in patients with early OSCCO. Overall, the results of our meta-analysis show that both amplification of CCND1 and overexpression of cyclin D1 correlate with nodal metastasis in OSCC. Furthermore, CCND1 amplification seems to have great potential as a diagnostic biomarker for lymph node metastasis in a subgroup of clinically nodal negative OSCC although supporting evidence is still not very strong.

The strength of a systematic review depends on the quality of the search, critical appraisal, and reporting of the review. For selection of studies, we used the validated QUADAS-2 tool to judge their quality [12]. The first finding in this critical appraisal was the large number of studies which included patients with head and neck cancer originating from different subsites, or used clinical nodal status as reference standard instead of histologically proven metastasis. Discrimination of head and neck subsites is particularly relevant, because multiple studies show differences in genetic alterations, such as mutations, amplification, or deletions, between its different subsites [34‐36]. As a consequence, the effects of amplification of 11q13 in any other location of the head and neck than the oral cavity cannot be extrapolated to OSCC. For this reason, we excluded all studies which included tumors other than OSCC and all studies without a separate location analysis. As mentioned earlier, the determination of nodal metastasis with imaging modalities is inaccurate in OSCC. Therefore, we excluded all studies using another reference standard than either histologically proven nodal metastasis or follow-up of the neck for at least 2 years [3].

Despite the fact that all studies analyzed included only OSCC and used histologically proven nodal metastasis as a reference standard, the correlation and diagnostic accuracy between amplification of the 11q13 region as well as overexpression of cyclin D1 and LNM still varied among the included studies. There are several possible explanations for these differences: First of all, there is heterogeneity in the stages of the included OSCCs. Most studies included all different stages of OSCC and three studies included only early stage OSCC [9, 26, 29]. Since early stages of OSCC show less genetic alterations than late stage OSCC, this could explain the stronger correlation in these three studies focused on stage 1–2 OSCC compared with the more variable correlation in studies that included all stages of OSCC [36]. Although Hanken et al. [22] found no significant differences in CCND1 amplification between T1-2 and T3-4 OSCC, this study did not look at the correlation of CCND1 amplification and LNM in these subgroups. Second, differences in methodological setup might explain part of the differences as the used assays, explored genes, and cutoff point for amplification varied between the 12 amplification studies. Although most protein expression studies used the same methods and explored the same genes (IHC for cyclin D1), these studies used different primary antibodies (see Table 4) and there was a wide range in the definition for overexpression (1–33 %) [8, 10, 27‐33]. Third, geographical or ethnical differences may account for a different outcome. Sixteen of the included studies were carried out in Asians, three in Caucasians, and one in an ethnically mixed group, although no divergent results were observed in outcome, which is in line with an earlier review [7]. Finally, one has to realize that OSCC includes tumors arising from different oral cavity subsites such as the cheek, floor of the mouth, and oral tongue. Meanwhile, an increasing number of studies have appeared that show differences in molecular biology between these oral cavity subsites [25] (Table 2).

This meta-analysis shows a significant correlation between both cyclin D1 protein overexpression as well as CCND1 amplification by FISH and the detection of nodal metastasis in OSCC. It is noteworthy that the strength of correlation between CCND1 amplification and the detection of LNM might be influenced by possible overlapping data in two included studies [9, 19]. Furthermore, a recent review in an Asian population by Zhao et al. found a slightly stronger correlation between cyclin D1 overexpression and nodal metastasis. However, they used a fixed-model method in their meta-analyses and also included studies using clinical nodal status as reference standard [7]. In order to use the fixed-model method, two conditions have to be fulfilled: (1) There must be good reasons to believe that all studies are functionally identical and (2) the computed effect cannot be generalized beyond the population included in the analysis. Although the Q-test for heterogeneity was not significant for cyclin D1 overexpression, we believe that the included studies are not functionally identical due to above mentioned differences in materials and methods, and therefore, we applied the more conservative random-model method. This model allows differences in effect size between studies and therefore leads to wider confidence intervals, especially if only few studies are included in the meta-analysis [15].

Meta-analysis of 11q13 amplification by CGH showed no significant correlation with nodal metastasis, which may seem contradictory to the other results. This inconsistency may be explained as follows: First of all, two studies (Yoshioka et al. and Pathare et al.) used relatively low cutoff values for amplification compared with the other CGH study and the FISH studies; see Table 2. Second, the total sample size of this meta-analysis was small and included only 176 patients, compared with 500 patients in the CCND1 amplification by FISH analysis and 813 patients in the cyclin D1 overexpression analysis; see Fig. 2.

For potential use as diagnostic tool in clinical decision making, the NPV of a biomarker in clinically lymph node negative OSCC is even more important than an overall correlation, since false negative results have serious consequences for the patient [37]. Unfortunately, only two studies (Myo et al. and Rodolico et al.) investigated the role of CCND1 amplification or cyclin D1 protein overexpression in this specific subgroup, both with a significant correlation with nodal metastasis [9, 30]. Of these studies, only Myo et al. reported sufficient data to reliably extract the NPV (83 %) and PPV (80 %). These results are promising considering the pre-test probability of 38 % for a nodal metastasis but need further validation in a larger cohort. The source data of the other study unfortunately could not be obtained from contacted authors.

Although we performed a comprehensive and systematic review with transparent methods, quality check, and extraction of study results, several limitations have to be mentioned. First, the search for this review was restricted to studies published in English, German, and Dutch, which after quality check led to inclusion of 20 articles (8 articles were excluded because of the language). In comparison with the review by Zhao et al., we could have missed some articles written in Chinese [7]. Second, because of the known inconsistence between clinically and histologically proven nodal metastasis, we only included studies that explicitly mentioned “pathological” or “histological” nodal metastasis in their manuscript and we left out three articles of moderate/good quality on risk on bias (at least three out of four items, low risk; see Table 1) that were unclear about their reference standard for nodal status. Although the likelihood of introduction of bias was minimized, potentially relevant studies could have been omitted from this analysis. Third, all presented studies are based on analysis of resection specimen. To be of diagnostic value for daily clinical practice, it would be relevant to validate these findings for incisional biopsies as well. Finally, we did not stratify our meta-analyses for anatomical subsites in the oral cavity. This may have to be taken into account in future analysis since evidence is increasing that these different locations might show different molecular alterations during carcinogensis [25].

In conclusion, according to current available evidence, both amplification of CCND1 and overexpression of cyclin D1 are potential biomarkers in the detection of LNM in OSCC. For early stage OSCC, which is the clinically most relevant subgroup, amplification of CCND1 had a NPV of 83 %. However, this evidence is based on only one study, and these results will have to be validated in a larger cohort of early OSCC, with subsite analysis. If these results confirm an association between CCND1 or 11q13 amplification and the presence of occult nodal disease in less than 20 %of the patients, this biomarker is of additional value in deciding as to whether or not treat the neck at early stage OSCC.