Novel Chromosomal Rearrangements and Break Points at the t(6;9) in Salivary Adenoid Cystic Carcinoma: Association with MYB–NFIB Chimeric Fusion, MYB Expression, and Clinical Outcome

Salivary adenoid cystic carcinoma (ACC), a relatively uncommon malignancy, is known for its progressive and heterogeneous clinical behavior (1–3). The primary treatment for patients with ACC is surgical resection with and without postoperative radiotherapy dependent upon the presence or the lack of adverse pathologic findings (4). More than 60% of these patients succumb to recurrent and/or metastatic disease with limited therapeutic options (4–6). Several recent genomic studies have attempted to unravel the events associated with ACC development and to identify molecular and biological markers for better management of patients with advanced disease (9, 12). Although no definitive marker(s) has been identified, recurrent loss of the terminal region of the long arm of chromosome 6 and translocation involving chromosomes 6q and 9p regions on different partners were the most consistently reported findings (7–13).

Recently, a fusion between the MYB and NFIB genes resulting from t(6;9)(q22-23;p24) regions have been identified in all 11 ACCs and were found to be associated with high 5′-MYB gene expression (14). Our group subsequently reported a lower incidence of MYB–NFIB fusion transcript, numerous fusion variants (15), and high level of the 5′- segment of the MYB transcript in the majority of fusion-positive tumors. We also noted that a subset of fusion-negative tumors express MYB level similar to those of fusion-positive tumors. These observations, together with the complex fusion variants and the high incidence of MYB–NFIB gene fusion by in situ hybridization (22), support the involvement of different molecular events associated with the t(6;9) in the MYB gene regulation (16–23) and other yet to be identified aberrations. We contend that accounting for these alterations is critical to understanding the role of the MYB–NFIB gene fusion in ACC development and progression.

To thoroughly account for the molecular genetic alterations associated with the t(6;9) in ACC and to understand their biological implications, we carried out detailed cytogenetic and molecular analyses on 30 new ACCs and the 52 previously screened MYB–NFIB fusion transcript–negative tumors and correlated the findings with the clinicopathologic parameters and the patient outcome.

We used fresh-frozen tissue specimens from 82 primary ACCs accessioned at the head and neck section from 1989 to 2010 which comprised 30 previously unanalyzed specimens and the 52 fusion transcript–negative tumors from our earlier report (15). Tumors were classified into tubular and cribriform if they lacked any solid component and manifested at 75% of either form (6). Tumors were categorized as solid if this feature is identified in any area. For clinical correlation of fusion-positive and fusion-negative tumors, we also included all fusion-positive tumors previously reported (15) for a combined total of 102 patients.

Total RNA was extracted with the TRIzol reagent (Invitrogen) and treated with recombinant DNase I, RNase free (Roche) prior to RT-PCR and converted subsequently to cDNA with the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) with oligo(dt) primers according to the manufacturer's instructions. The amplification of the MYB–NFIB fusion transcripts and the primers used were described previously (Supplementary Table S1; ref. 15). The MYB–NFIB fusion transcripts were detected by PCR analysis with Platinum Taq DNA polymerase (Invitrogen). We designed a new set of primers (Supplementary Table S1) in addition to the previously published primer sets (Supplementary Table S1). ACTB primer (Supplementary Table S1) was used as internal control. RT-PCR products were purified and sequenced directly or cloned into the pCR2.1 vector (Invitrogen). The PCR fragments were sequenced with an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) at the DNA sequencing core facility, MD Anderson Cancer Center. The MYB–NFIB variants, the nucleotide sequences, and genomic organization of MYB (accession number NM_001130173) and NFIB (ENSG00000147862) were determined by the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) and Ensemble (http://ensembl.org), respectively.

Quantitative RT-PCR was carried out with the 7900HT Fast Real-time PCR systems (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems). The primer sequences are shown in Supplementary Table S1. The ACTB gene was used as internal control. Duplicate samples for each tumor or tissue were analyzed. The expression of MYB transcripts was determined by the ΔC_t method (Average CT-MYB- Average CT-ACTB), and relative MYB expression in each tumor was determined on the basis of MYB expression in a pooled normal salivary gland standard (Clontech Laboratories). The average of each individual tumor and this value was used to determine the average for fusion-positive and fusion-negative tumor groups. In this study, we considered the upper quartile of MYB values to represent the high expression.

FISH was conducted on touch preparations of ACCs to identify MYB/NFIB rearrangements. We initially used bacterial artificial chromosome (BAC) clones containing MYB gene (RP11-104D9) and NFIB gene (RP11-54D21 and RP11-79B9) for screening of the gene fusion (Fig. 1A). The probes were labeled with Spectrum Green and Spectrum Red, respectively (Abbott Laboratories). Hybridization and detection were conducted according to the manufacturer's protocols. Two hundred individual nuclei were analyzed for each case, and the interphase nuclei was captured and processed with the Quantitative Image Processing System (Applied Imaging). In nuclei containing the MYB–NFIB fusion, green and red signals from the MYB and NFIB genes overlap in a red/green (yellow) signal.

To localize the alternate break points in fusion-positive transcript–negative tumors, we selected 2 BAC clones; one at 5′ of MYB (RP11-378M4) and the other at 3′ of MYB gene (RP11-55H4). Both of these clones overlap with the MYB clone used in the first FISH (RP11-104D9) screening probe (Fig. 3A).

To evaluate the 3′ end of MYB transcript sequence or detect the unknown gene fusion of MYB, first-strand cDNA was synthesized from 2 μg of total RNA by M-MLV reverse transcriptase (Ambion) using 3′RACE adapter primer (Invitrogen; Supplementary Table S1). 3′RACE nested PCR was done with 2 sets of MYB gene–specific primers (Supplementary Table S1) and 3′RACE universal primer (AUAP; Invitrogen; Supplementary Table S1) to generate a specific amplification product. PCR products were purified and sequenced directly or cloned into the pCR2.1 vector (Invitrogen) and were then sequenced.

Genomic DNA was extracted with the Gentra Puregene Tissue Kit (QIAGEN) according to the manufacturer's instructions. DNA copy number from 4 tumors and corresponding normal specimens was analyzed by affymetrix GeneChip Human Mapping 250k NSP array. The mapping information of the single-nucleotide polymorphism (SNP) sites was provided by the human genome sequence version NCBI36/hg18. Array data analyses were conducted with Partek software and R packages.

The procedures for genome wide paralleled paired-end sequencing to identify somatic genomic alterations and rearrangements in tumor specimens were carried out as previously described (24, 25). Briefly, 5 μg of genomic DNA from tumor and normal specimens were sheared to 400 to 500 bp fragments. Sequencing of 37 bp from either end was carried out on the Illumina Genome Analyzer II platform. Reads were aligned to reference human genome (NCBI build 36) using MAQ (26), with a coverage up to 1-2X sequence (50–60 million reads, 37 bp paired and N400 6p inserts), giving a physical coverage of 6-8X (25). Putative genomic rearrangements were screened by PCR across the break point in tumor DNA samples and germ line DNA (27).

Descriptive statistics for scaled values and frequencies of study patients within the categories for each of the parameters of interest were enumerated with the assistance of commercial statistical software. Correlations between categorical parameters and endpoints were assessed by Pearson χ² test or, where there are fewer than 10 subjects in any cell of a 2 × 2 grid, by the 2-tailed Fisher exact test. Because the values for expression of MYB at exons 2–3 and at exons 15–16 did not meet tests for normality, possible differences between groups were assessed by the nonparametric Mann–Whitney U test. Curves describing overall survival were generated by the Kaplan–Meier product limit method. The statistical significance of differences between the actuarial curves was tested by the log-rank test. Follow-up time was the time from first appointment at the University of Texas MD Anderson Cancer Center for the primary tumor of concern until the date of last contact or death. Proportional HRs and multivariate models were assessed by Cox regression analysis. These statistical tests were conducted with the assistance of the Statistical (StatSoft, Inc.) and SPSS (IBM SPSS, IBM Corporation) statistical software applications.

In the initial phase of the study, we analyzed 30 new ACCs representing equal number of patients by multiple complementary techniques to account for heterogeneity of the molecular genetic alterations associated with the t(6;9). The patients comprised 17 males and 13 females who ranged in age from 39 to 94 with a mean of 62 years. The tumor size ranged from 0.2 to 12.0 cm, with mean of 3.1 cm and 20 tumors (66.7%) had perineural invasion. All patients underwent surgical resection with curative intent and postoperative radiotherapy. Table 1 presents the genetic and molecular findings of 30 new ACCs by different techniques.

To screen for the MYB–NFIB gene fusion and the transcript formation, we conducted FISH with BAC clones for the MYB gene (green, RP-11-104D9, Fig. 1A) and the NFIB gene (red, RP11-54D21 and RP11-79B9, Fig. 1A) and RT-PCR using the original set of and newly designed primers on all 30 tumors (Fig. 1B) and all fusion PCR products were sequenced. None of the FISH-negative tumors had fusion transcripts formation by RT-PCR. FISH analysis showed 17 (56.7%) tumors to be positive for the MYB and NFIB genes translocation (Table 1). Nine (52.9%) of the FISH-positive samples had detectable fusion transcript and 8 (26.7%) lacked transcript product (Fig. 1C and Table 1); these data suggest that additional rearrangements or break points other than MYB–NFIB fusions are present. Considering the complex and evolving information on the structural formation of the MYB gene, (28) we observed that exon 10 of the gene is lost in all sequenced MYB–NFIB chimeric transcript and the intact form of MYB transcript in ACC. This exon is also known as exon 9B, using NCBI accession #HSU22376.

To assess alteration at the 3′end of the MYB transcript, 3′RACE amplification was carried out and led to the identification of 3 fusion transcripts; these were not detected by RT-PCR analysis (Table 1). Interestingly, 2 FISH-positive tumors (#290F8 and 394D7, Fig. 1D) had fusion transcripts comprising MYB exon 15 and the 3′ untranslated region (UTR) of the NFIB gene and manifested the expected high level of MYB-truncated transcript (Fig. 1E and Supplementary Fig. S1); 1 tumor (# 288F7) had fusion between MYB exon 13 and intron 22 of the EFR3A gene on chromosome 8q24 (Table 1 and Supplementary Fig. S2); in addition to a stop codon at intron 13 and MYB-truncated transcript.

We previously reported loss or marked reduction of the full-length MYB (exon 15–16) transcript expression in all MYB–NFIB fusion-positive tumors. To confirm this finding, we analyzed the expression levels of MYB transcripts in 30 salivary tumors by quantitative RT-PCR with primers for MYB exons 2–3 and the last MYB exon. Overall, the expression of MYB exon 2–3 in fusion transcript–positive tumors (average 361) was more than 2-fold higher than the majority of their fusion transcript–negative ACCs (average 172; Table 1). The analysis also shows that the expression levels of MYB exon 2–3 and the last exon were comparable in the majority of tumors with genomic MYB–NFIB fusion without transcript formation. In 1 tumor (#288F7, Table 1) with MYB and EFR3A gene fusion, the expression of the last MYB exon was markedly reduced whereas the 5′-segment was moderately elevated. We also observed that the 3 tumors with the highest 5′-segment MYB transcript (cases # 404D3, 369B5, and 133C4) had fusion between MYB exon 8b and exon 12 of NFIB gene (Fig. 1F and Table 1).

On the basis of these findings, we extended our analysis to include the 52 MYB–NFIB fusion transcript–negative ACCs from our previous study (15). The analysis showed that 17 (32.7%) of the 52 tumors to be positive for gene fusion by FISH, and only 8 of these (6 by PCR and 2 by the 3′RACE) were MYB/NFIB transcript–forming tumors (Table 2). Two of these tumors (542A1 and 318H3) had fusion between MYB exon 14 (Supplementary Fig. S3) and intron 11 (Supplementary Fig. S4) and the NFIB 3′UTR, respectively. In addition, we identified a novel t(6;9) rearrangement involving MYB exon 15 and an inverted sequence of the PDCD1LG2 intron 3 (Table 2 and Supplementary Fig. S5) in another tumor (# 405B2). Another tumor (485F7) showed MYB–NFIB gene fusion by FISH but no transcript formation or MYB alterations was found (Fig. 2A).

To account for the translocation sites in gene fusion-positive but fusion transcript–negative ACCs, we selected additional BAC clones at the 3′ and 5′ of the MYB gene that overlaps with the original MYB probe (Fig. 3A). The results revealed, 5′ fusion signal proximal to the MYB gene in 1 tumor (Supplementary Table 2), a positive 3′ probe signals in 8, and fusion signals were found for both the 3′ and the 5′ flanking probes of the MYB in 2 tumors. Surprisingly, the 2 cases (288F7 and 405B2) in which MYB fused with EFR3A or PDCD1LG2 showed as a positive by 3′ probe signal suggesting that each allele fused different genes. These findings localize the break points distal to the 3′ end of the MYB gene in 8 tumors and to the proximal 5′ end of the gene in 1 tumor. In the 2 tumors positive for both probes, either reciprocal translocation and/or translocation in 1 allele and insertion involving the other allele may have occurred. Detailed molecular analyses of these 2 tumors are underway.

To survey the genomic findings in tumors representing the MYB–NFIB gene fusion status, 4 tumors [2 fusion negative, 1 FISH positive/transcript negative (485F7, Supplementary Fig. S6), and 1 fusion positive by both FISH and RT-PCR (325E5, Supplementary Fig. S6)] were analyzed by massively pair-ended sequencing to confirm the molecular results and to screen for new alterations. The analysis confirmed the lack of any abnormalities in the 2 fusion-negative tumors and validated the presence of MYB–NFIB gene fusion in the fusion-positive tumor. In the FISH-positive/transcript-negative tumor (#485F7, Fig. 2A), complex alterations were observed; Fig. 2B represents schematic illustration of the inter- and intrachromosomal changes in this tumor; these included a breakage and translocation of intron-7 sequence of the NFIB to a 99 kb upstream location of the MYB coding region (Supplementary Table S3) and a novel fusion between NFIB gene and 2 alternative variants of the AIG1 gene on chromosome 6q24. The NFIB-AIG1 fusion transcript resulting from this translocation was confirmed by RT-PCR and sequencing analyses (Supplementary Fig. S7).

The SNPs copy number analysis of chromosomes 6 and 9 using same 4 ACCs are shown above and below the schematic illustration in Fig. 2B. Comparison of the massively parallel paired-end sequencing (Supplementary Fig. S6) and Affymetrix 250 k SNP genomic array data confirmed that the genomic alterations occurred at copy number neutral region of chromosome 6 (Fig. 2B and Supplementary Fig. S8).

The combined analysis of the 102 (30 new and 72 previously reported) tumors showed that 54 (52.9%) had genomic MYB–NFIB gene fusion, 39 (38.2%) of these formed fusion transcript, and 48 (47.1%) were negative for any fusion-related alterations (Supplementary Table S4). The expression of both MYB exon 2–3 and exon 15–16 segments in transcript forming tumors was significantly (Mann–Whitney U test, Supplementary Table S4) higher than those with transcript negative tumors (P < 0.001 and 0.003, respectively). The expression of MYB exon 2–3 in tumors with only genomic fusion (by FISH) was significantly higher than in fusion transcript–negative tumors (P < 0.001). The expression level of MYB transcript (exon 15–16) in fusion transcript–negative tumor was not significantly different from the expression of gene fusion-negative tumors (Supplementary Table S4).

We further examined the association of tumors with MYB–NFIB fusion with and without transcript formation and level of MYB expression (exon 2–3) and the clinicopathologic factors and patients outcome. An arbitrary cutoff level of 470 for MYB expression was based on the upper quartile of MYB (exon 2–3) expression among all the samples tested. This value was used in the statistical correlative analysis. Kaplan–Meier analysis showed significant correlation between high MYB expression and poor survival (P = 0.004, log-rank test Fig. 4A). Univariate Cox proportional hazard regression analyses showed that high MYB expression, age of 60 years or more, and tumor with solid component were significant prognostic factors (P = 0.005, 0.008, and <0.001, respectively, Table 3). Interestingly, significantly different survival plot for patients with low MYB and solid tumors with those who had high MYB and solid phenotype tumors was found suggesting that high MYB expression correlates with poor outcome independent of the solid phenotype (Fig. 4B).

Our study identified novel and a spectrum of complex cytogenetic and molecular alterations associated with the t(6;9) event in ACC. The results show that approximately 53% of ACCs showed genomic MYB–NFIB fusion with and without fusion transcript formation. These findings are in agreement with those recently reported in a retrospective study of this entity (22). The majority of tumors with genomic fusion represented in-frame translocation of the MYB and the NFIB genes with the formation of variable chimeric fusion transcripts in a cell- and tissue-specific context (15). Interestingly, in the subset of nontranscript forming MYB–NFIB gene fusion tumors, we identified translocation break points at the flanking sites of the MYB gene on chromosome 6q24 region with no evidence of MYB transcript alteration. Similar break points at the flanking sequences of fusion genes in several neoplastic entities have also been reported (16, 17). The predicted biological consequences of these alterations are most likely the disregulation of critical oncogenes neighboring these sites (29–32).

In this study, multiple break points exclusive of those reported (16, 33, 34) between the MYB and the NFIB genes were identified. These included translocations involving, exon 15 of MYB and intron 3 of the PDCD1LG2 gene on chromosome 9p24 and MYB exon 13, intron 22 of the EFR3A gene on chromosome 8q24, and the NFIB with the AIG1 on chromosome 6q24. In addition, a separate intragenic translocation of an NFIB sequence to a proximal site of the MYB coding region was also identified in the latter tumor. The translocation involving intron sites in these instances have previously been reported in a benign [HMGA2 and COG5 (35) NFIB and to the HMGA2 gene (36–40)] and malignant tumors (40, 41). The mechanistic association of these uncommon events in the oncogenesis of ACC, however, remains to be elucidated.

Our findings strongly link the MYB–NFIB gene fusion to the upregulation of the MYB gene in ACCs. This may likely be due either to the lack of the 3′UTR, which includes the regulatory microRNA target sites, or to the deletion of the terminal negative regulatory domain of the MYB fusion transcript–positive ACCs. The translocation of other genetic sequences to the noncoding flanking sites of the MYB gene can also lead to the MYB transcriptional activation perhaps through epigenetic modification including histone acetylation especially in MYB–NFIB fusion transcript–negative tumors (19, 21, 23, 34, 43). Other upstream events affecting the transcription of the MYB gene in fusion transcript–negative tumors including (43) the NFIB sequence translocation upstream of the coding region leading to high MYB expression can be involved. Interestingly, our findings are distinctly different from those associated with the activation of this gene in other solid tumors including colon (19, 36, 44) and breast carcinomas and suggest that MYB regulation varies in a tissue- and tumor-specific context (33, 34, 37, 38, 42, 45–48).

In this study, clinicopathologic analysis identified 3 factors including patients older than 60 years, solid phenotype and high MYB expression to be significantly associated with poor survival. In both, multifactorial Cox HR, log-rank testing and the survival plot for patients with low MYB and solid type was significantly different from patients with high MYB and solid type. We contend, however, that further studies are required to assess the functional threshold of MYB expression, to identify chimeric fusion protein and to determine the significance of the selective MYB expression to myoepithelial cells in the pathobiology of ACC. These studies will be further advanced by the availability of reagents that distinguish between the MYB protein variants as well as results from second generation deep sequencing of these tumors. Overall, the data indicates that the juxtaposition of the terminal sequences of the NFIB within and around the coding MYB gene sequence is the main genetic event in the t(6;9) positive ACCs and this leads to an elevated 5′ segment of the MYB gene in ACC (31, 32, 49).

In conclusion, we comprehensively accounted for the alternative MYB–NFIB gene fusions and the other molecular genetic alterations associated with the t(6;9) in ACCs and showed that marked genetic heterogeneity are associated with this event. The study characterized 2 main events resulting from the t(6;9): one with gene fusion alone and another with gene fusion and chimeric transcript formation. The former is due to break points at the flanking sites of the MYB gene. These alterations require multiple methodologic approaches for their detection. The results also show that transcript forming ACCs express high 5′-truncated MYB segment and pursue aggressive behavior. We, therefore, contend that the translocation of the NFIB terminal sequences as a result of the t(6;9) and different molecular events, may underlie the transcriptional regulation of the MYB gene in ACC.

No potential conflicts of interest were disclosed.

The authors are grateful to Dr. Scott A. Ness for update information on the MYB gene and to Deborah A. Rodriguez and Stella U. Njoku for technical and Wendy Garcia for secretarial assistance.

The study was supported, in part, by the NIH National Institute of Dental and Craniofacial Research (NIDCR) and the NIH Office of Rare Diseases Research (ORDR) grant number U01DE019765, the Head and Neck SPORE program grant number P50 CA097007, The Kenneth D. Muller professorship and the NCI-CA-16672 grant. P.A Futreal and P.J. Stephens acknowledge the support of the Wellcome Trust under grant reference number 077012/Z/05/Z. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the NIH. The Adenoid Cystic Carcinoma Research Foundation (ACCRF).

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