Amplification and co-amplification of LANCL2 and EGFR were prevalent in glioblastoma, but were not the independent prognostic markers for IDH1/2-wild-type glioblastoma patients.
Firstly, to investigate the CNVs of LANCL2 and EGFR genes in a panel of cancers, 32 studies of different cancer types in TCGA Pan-Cancer Atlas database (n=10967) were selected. Results showed that the dominant genomic alterations of LANCL2 and EGFR in cancers were amplification and mutation, while gene fusion and deep deletion were rare. Glioblastoma, head and neck squamous cell carcinoma, esophagogastric adenocarcinoma and non-small cell lung cancer were the top four tumors with the highest alteration frequencies of LANCL2 and EGFR (Fig.1A-B). Subsequently, two studies Glioblastoma Multiforme (n=592) and Brain Lower Grade Glioma (n=514) were further analyzed. The amplification frequencies of LANCL2 and EGFR in GBM were up to 27.65% (159 of 575 cases) and 44.35% (255 of 575 cases), whereas those in LGG were only 3.91% (20 of 511 cases) and 7.63% (39 of 511 cases), respectively (Fig.1C). The data of LGG contained grade II and III gliomas, including oligodendroglioma, oligoastrocytoma and astrocytoma. Among the LGG data, the amplification frequencies of LANCL2 and EGFR in astrocytoma were the highest (7.33% and 13.92%, respectively), while those in oligoastrocytoma were the lowest (1.07% and 1.60%, respectively) (Fig.1D). Co-amplification of LANCL2 and EGFR was common in GBM, but it was rare in LGG. LANCL2 amplification was found in 61.96% of GBM samples and 51.28% of LGG samples containing EGFR amplification. Furthermore, nearly all GBM and LGG samples containing LANCL2 amplification displayed EGFR amplification (Fig.1E). The main types of LANCL2 and EGFR CNVs in GBM were copy number gain and amplification, whereas shallow deletion and diploid were infrequent. Chi-square test demonstrated a significant correlation between the CNVs of LANCL2 and EGFR (p<0.001) (Fig.1F). We analyzed the top ten genes which had the highest co-amplification frequencies with LANCL2 or EGFR. Results indicated that the amplification frequencies of EGFR, SEC61G and VOPP1 genes were the top three highest in LANCL2-amplified GBM samples, while SEC61G, LANCL2 and VSTM2A were the top three genes co-amplified with EGFR (Fig.1G-H). Kaplan-Meier survival analysis was performed to evaluate the prognostic values of LANCL2 and EGFR amplification in GBM patients. Results showed that LANCL2 and EGFR amplification was significantly associated with reduced OS but not PFS of patients with GBM (Fig.1I-L).
The relationship between CNVs of LANCL2/EGFR and molecular pathology of GBM samples was analyzed. Wild-type IDH1/2 was mainly found in GBM samples with LANCL2/EGFR gain or amplification. Chi-square test found that CNVs of LANCL2 or EGFR were significantly correlated with IDH1/2 mutation but not MGMT methylation status (Fig.2A-D). To observe whether the prognostic value of LANCL2/EGFR amplification was affected by IDH1/2 mutation status, Kaplan-Meier survival analysis was performed in IDH1/2-wild-type GBM patients (n=354). Results found that LANCL2 or EGFR amplification was not significantly correlated with OS and PFS of IDH1/2-wild-type GBM patients (Fig.2E). Similarly, co-amplification of LANCL2 and EGFR was also significantly associated with reduced OS but not PFS of GBM patients. However, in GBM patients with wild-type IDH1/2, co-amplification of LANCL2 and EGFR was not correlated with OS and PFS (Fig.2F).
mRNA overexpression of LANCL2 and EGFR were frequent in glioblastoma, but were not associated with the prognosis of glioblastoma patients.
The mRNA expression profiles of LANCL2 and EGFR were investigated in 32 different cancers of TCGA database. In the histograms, the average mRNA expression of LANCL2 and EGFR was organized from lowest to highest priority. Among them, LGG, testicular germ cell carcinoma, GBM and uveal melanoma were the top four tumors with the highest average mRNA expression of LANCL2, while the average mRNA expression of EGFR was highest in GBM, head and neck cancer, clear cell renal cell carcinoma (ccRCC) and LGG (Fig.3A-B). mRNA overexpression of LANCL2 and EGFR was found in 35.63% (57 of 160 cases) and 48.13% (77 of 160 cases) of GBM samples, respectively (Fig3.C). However, the mRNA overexpression frequencies of LANCL2 and EGFR in LGG samples were only around 10%, and little difference was shown in astrocytoma, oligoastrocytoma and oligodendroglioma (Fig.3D). The correlation between mRNA expression and CNV of LANCL2 and EGFR was then analyzed. Results showed that mRNA expression of LANCL2 was significantly elevated in GBM samples with LANCL2 amplification, compared with GBM samples with diploid or gain of LANCL2 (Fig.3E). Likewise, the correlation was the same in EGFR (Fig.3F). However, mRNA overexpression of LANCL2 or EGFR was not significantly associated with OS and PFS of GBM patients (Fig.3G). Moreover, concurrent mRNA overexpression of LANCL2 and EGFR was found in 26.25% (42 of 160 cases) of total GBM samples and 54.55% (42 of 77 cases) of EGFR-overexpressed GBM samples (Fig3.H). In addition, linear regression analysis demonstrated that mRNA expression of LANCL2 and EGFR was positively correlated (p<0.001)(Fig.3I). Nevertheless, concurrent overexpression of LANCL2 and EGFR was also not significantly correlated with OS and PFS of GBM patients (Fig.3J-K). Interestingly, mRNA expression levels of EGFR were significantly elevated in IDH1/2-wild-type GBM samples, while no obvious change of LANCL2 mRNA expression was found, suggesting a significant association between EGFR mRNA expression and IDH1/2 status (Additional file: Figure S1A-B). However, mRNA overexpression of LANCL2 or EGFR was also not significantly associated with OS and PFS of IDH1/2-wild-type GBM patients (Additional file: Figure S1C-D).
CNVs, mRNA expression and the prognostic values ofLANCL2 and EGFR in low-grade gliomas
To evaluate the prognostic values of LANCL2 and EGFR CNVs and mRNA expression in LGG, the study of LGG (n=514) was analyzed. The dominant type of LANCL2 and EGFR CNVs in LGG was diploid, and chi-square test revealed that the CNVs of LANCL2 and EGFR were significantly correlated (p <0.001)(Fig.4A). The mRNA expression profiles of LANCL2 and EGFR were then investigated, showing that LANCL2 and EGFR mRNA expression was significantly increased when these genes amplified. However, no difference among LGG samples with shallow deletion, diploid and gain of LANCL2 or EGFR was observed (Fig.4B-C). In addition, mRNA expression level of LANCL2 were also positively correlated with that of EGFR in LGG samples (Fig.4D). Similar as the GBM samples, CNVs and mRNA expression of LANCL2 or EGFR were significantly correlated with IDH1/2 mutation status (Fig.4E-H). All patients with LANCL2 or EGFR amplification contained wild-type IDH1/2. These indicated that LGG patients’ survival may also be affected by the IDH1/2 status. Therefore, we evaluated the prognostic values of LANCL2 and EGFR CNVs and mRNA expression in IDH1/2-wild-type LGG patients. Unfortunately, results also showed that amplification or mRNA overexpression of LANCL2 and EGFR was not significantly correlated with OS and PFS of IDH1/2-wild-type LGG patients (Fig.4I).
Amplification and co-amplification LANCL2 and EGFR were also frequent in glioblastoma from the tumor banks, and were associated with poor overall survival of glioblastoma patients.
To validate the analysis results of TCGA database, we analyzed the copy numbers of 100 GBM patients’ samples from our tumor banks by Taqman Copy Number Assay using fluorescent probes targeting LANCL2 and EGFR. The log2 copy number values larger than 2 was regarded as amplification. Results showed that compared with the copy numbers in normal brain tissues and grade I gliomas, the copy numbers of EGFR were significantly elevated in GBM, while the copy numbers of LANCL2 had no obvious changes (Fig.5A-B). Interestingly, when the GBM samples were subdivided into newly diagnosed and relapsing tumors, the copy numbers of LANCL2 and EGFR were significantly increased only in newly diagnosed GBM (Fig.5E-F). The amplification frequencies of LANCL2 and EGFR were 62.00% and 55.00% in 100 GBM patients, respectively (Fig.5C, Table.1). LANCL2 and EGFR co-amplification was found in 47.00% of the total GBM samples and 85.45% of GBM samples containing EGFR amplification (Fig.5D, Table.1). Pearson’s correlation analysis also showed that the copy numbers of LANCL2 and EGFR were positively correlated with each other (Fig.5G). Chi-square tests showed that amplification of LANCL2 was not correlated with IDH1 and TERT mutations, and MGMT methylation, whereas amplification of EGFR was significantly associated with IDH1 and TERT mutations. On the other side, co-amplification of LANCL2 and EGFR was not related with TERT mutation and MGMT methylation, but was correlated with IDH1 mutation (Fig.5H). Without regard to the influence of IDH1 status, LANCL2 or EGFR amplification, and their co-amplification were significantly associated with decreased OS of GBM patients (n=81) (Fig.5I). However, LANCL2 or EGFR amplification, and their co-amplification were not correlated with OS of IDH1-wild-type GBM patients (n=20) (Fig. 5J).
Protein expression and localization of LanCL2 was independent to EGFR in gliomas
To investigate the protein expression profiles of LanCL2 and EGFR, 72 GBM samples (including newly diagnosed and relapsing tumor samples) and 4 low-grade (grade I) glioma samples from our tumor banks were used. Compared with the grade I glioma control, the log2 relative protein expression values larger than 2 was regarded as overexpression. We found that overexpression of LanCL2 and EGFR was found in 38.89% and 58.33% of the total GBM samples (Fig.6A, Table.2). The protein expression of EGFR was markedly increased in GBM samples, whereas the expression levels of LanCL2 had no significant change (Fig.6B-C). Interestingly, overexpression of LanCL2 was observed in relapsing GBM compared with newly diagnosed GBM (Fig.6D, F). On the other hand, although both the newly diagnosed and relapsing GBM samples displayed elevated EGFR expression compared with the grade I glioma samples, no significant change was found between the newly diagnosed and relapsing GBM samples (Fig.6E-F). Pearson’s correlation analysis showed that the expression levels of LanCL2 and EGFR were not correlated (Fig.6G). Chi-square tests showed that overexpression of LanCL2 or EGFR was not significantly associated with IDH1 or TERT mutations, and MGMT methylation (Fig.6H). No significant association was also found between the expression of LanCL2/EGFR and OS of GBM patients (Fig.6I-J). Subsequently, we used tissue microarray to investigate the expression pattern of LanCL2 and EGFR in GBM cells. Results also showed that the expression scores of both LanCL2 and EGFR were markedly increased in GBM tissues, compared with normal brain tissues (Fig.7B, D). LanCL2 was expressed in both the normal brain tissues and gliomas. The protein expression level and intracellular localization of LanCL2 were correlated with the grade of gliomas. The higher the glioma grade, the higher the expression intensity of LanCL2. LanCL2 was mainly found in the nucleus and cytoplasm of high-grade glioma cells (grade III-IV), whereas it was expressed on the nuclear membrane of low-grade (grade I-II) glioma cells (Fig.7A). On the other hand, EGFR was barely expressed in the normal brain tissues and low-grade gliomas, but was overexpressed in the grade III-IV gliomas. It was mainly located in the plasma membrane and cytoplasm of both low-grade and high-grade glioma cells (Fig.7C).