The clinical characteristics of 31 solid type ACC cases
The clinical pathologic characteristics and follow-up information of 31 solid type ACC patients (solid type component >30%) are presented in Table 2. The mean age at presentation was 56.1 years (range: 31 to 89 years) with a male predominance (male to female ratio of 2.4:1). The solid type ACC cohort consisted of 26 tumors that were predominantly solid pattern (more than 80%) and 5 tumors (ACC4809, 4802, 4810, 4813 and 4806) that comprised a roughly equal mixture of cribriform/tubular and solid histological forms. These tumors, prone to developing at the maxilla (13/31, 42%), were usually grossly aggressive with extraglandular extension from maxilla sites and with bone involvement at sinonasal or palatine sites. The maximum tumor diameter ranged from 1.2 to 8 cm with a median size of 4cm. Neck dissections were performed in addition to surgical excision in 3 cases when imaging tests indicated lymph node metastases. Distant metastases were clinically noted in sixteen patients, though only 1 case with lung metastases was biopsy proven. Focal necrosis was present in 20 cases (20/31, 64.5%). 16 tumors presented perineural invasion (16/31, 51.6%) and 9 tumors had vascular invasion (9/31, 29.0%). The leading cause of death in these cases was local relapse with skull base infiltration.
The genetic profiles of solid type ACC
To further delineate the molecular profile of solid type ACC, a comprehensive molecular analysis of solid components microdissected from 31 solid type ACCs was performed. A total of 316 genomic alterations (307 mutations + 9 copy number alterations) were identiﬁed in the solid components of 31 tumors with an average of 10.2 per tumor. A total of 110 genomic alterations (103 mutations + 7 copy number alterations) with potentially actional significance were included, in which 30 of the 31 tumors (96.8%) harbored at least one with an average of 3.7.
Several genes were identified in which the alterations were significantly enriched in the solid type ACCs (Figure 1A) and lollipop plots of key mutated genes were shown in Figure 1C. NOTCH1, a critical regulator of cell fate determination and various developmental processes, was the most frequently mutated gene in 21/31 (67.7%) cases. Forty somatic mutations in NOTCH1 were found including 16 frame-shifts, 6 in-frame insertions/deletions, 14 missense mutations and 4 nonsense mutations. 14 samples (13/21, 61.9%) displayed multi-hit somatic mutations to the NOTCH1 gene, of which 13 samples were predicted to be NOTCH1 activating, in keeping with its potential oncogenetic role. Thirty-one of these NOTCH1 genetic alterations occurred in the extracellular domain, heterodimerization domain (HD) and PEST domain that have been identified in multiple cancer types including T-cell acute lymphoblastic leukemia, leading to gamma-secretase inhibitor (GSI) sensitivity. This finding suggests that they were gain-of-function mutations. The four nonsense mutations were all predicted to generate premature stop codons that could result in truncation mutations and disrupt the PEST domain, causing a modest increase in activity. Another NOTCH1 alteration in Patient 1959 that disrupted ligand binding led to the loss of the ankyrin repeats, which are necessary for NOTCH1 function and predicted to be inactivating. The four other NOTCH1 mutations in the EGF-like extracellular domain of the NOTCH receptor have not been well-characterized, and their effect on function is unclear, although similar alterations have been reported in the context of cancer, which may indicate biological relevance. Furthermore, another member of the NOTCH pathway, SPEN, which was found to repress the transcriptional activity of NOTCH as a tumor suppressor, was mutated in 7/31(23%) cases. Other members of the NOTCH family with a lower prevalence of alteration were also observed: NOTCH3 (4 cases, mutation, 11.1%) and NOTCH2 (1 mutation, 2.8%). Generally, approximately 77.4% (24/31) of cases harbored an alteration in the broader NOTCH pathway, including FBXW7. Mutations in genes important in histone modification and chromatin-remodeling were also significantly enriched in solid type ACC, including CREBBP (11 cases, 35%), EP300 (9 cases, 29%), KDM6A (8 cases, 26%) and MLL2 (6 cases, 19%). BRCA2 mutations, which confer sensitivity to PARP inhibitors, were detected in 23% (7/31) of solid type ACCs, but were quite uncommon in the cribriform/tubular type.
Moreover, ten copy number alterations (CNAs) were detected, including losses involving 5q and 18q and gains involving 1p, 8q and 10q. On chromosome 1p, the locus of the NOTCH2 gene (1p12), the HSD3B1 (1p12) and the MYCL1 gene (1p34.2) showed an amplification in 6% (two cases) of the solid type ACCs. Notably, two patients with NOTCH2 amplification were not only likely to develop lung metastasis, but also had a far higher likelihood of developing metastasis in the liver. Another region of interest on chromosome 8q is the locus of the MYC and RAD21 genes (8q24), which showed an amplification in 6% (two cases) of solid type ACCs. Region 5q11.2, where the MAP3K1 gene is located, showed a loss in 1 case. The chromosome 18 where contained the SMAD4 gene was deleted in 1 case as well (Supplementary Figure 2).
We then focused on the status of fusion genes identified in solid type ACCs. MYB-NFIB fusion was detected in 6 cases (6/31, 19%) of the solid type and demonstrated that they all involved MYB exon 14 and NFIB exon 9. In Patient 1955, rearrangements of MYB with APOO were found. Additionally, MYB truncation was identified in Patient 4819. Collectively, MYB rearrangement may not be the key genetic alteration in solid type ACC, although substantial complexity of the structural rearrangements in the MYB gene was indicated.
Heterogeneity of molecular alterations in the distinct components within solid type ACCs
Intratumor heterogeneity, leading to variable treatment responses, has been one of the major challenges in treating cancer. Comprehensive analysis of multiregional samples aids in understanding intratumoral heterogeneity. To this end, the consistency and discrimination of genetic alterations between the cribriform/tubular component and solid components microdissected separately within a high-grade tumor were assessed in 5 cases (ACC4809, 4802, 4810, 4813 and 4806) (Figure 2A).
Phylogenetic tree analysis demonstrate that all 5 ACCs had some genetic alterations that were common among other histological components but with an enrichment in the cancer cell fractions from the cribriform component to the solid component and other alterations that were component-specific. In ACC 4813, two frame-shift mutations in NOTCH1 (R1594fs and P1582fs) and one missense mutation in MRE11A (P629S) were restricted to solid type ACC, with cell fraction of 24%, 5% and 41% respectively. Mutations in NOTCH1 (P1582fs, 5%) were subclonal, indicating the presence of intratumor genetic heterogeneity. In addition, the nonsense mutation in CSF3R (Q707*) became clonal in the solid type ACC, with cancer cell fractions ranging from 40% in the cribriform/tubular ACC to 90% in the solid type ACC (Figure 2B ,2C and 2D). ACC 4809 is a tumor derived from the sweat glands of skin near the nose. Three nonsense mutations in genes EP300 (Q341*), SPEN (R702*) and HSD3B1(R250*) existed in both components of ACC4809, but there was an enrichment in the cancer cell fractions from 8%, 14% and 74% in the cribriform component to 45%, 76% and 88% in the solid component respectively. A frameshift in NOTCH1 (F1736fs) was detected in the cribriform component and missed in the solid component, whereas the copy number amplification in NOTCH2 was acquired in the solid component (Figure 2B ,2C and 2D Supplementary Figure 2). In ACC 4810, A frame-shift mutation in MLL2 (R2188fs) and a missense mutation in PPP2R2A (R252Q) were restricted to the solid component with the cancer cell fractions of 32% and 38% respectively (Figure 2B and 2C). In ACC 4806, a missense and subclonal nonsense mutation in TP53 (C275Y and R342*), was restricted to cribriform ACC. Akin to ACC 31, the missense mutation in NOTCH1 (L1709P) was acquired in the progression to solid type ACC with a cancer cell fraction of 62% (Figure 2B and 2C). In ACC 4802, 1 splice site mutation in KDM6A (splice site 564+1G>C) and 3 missense mutations in PIK3C2G (R781C), PTCH1 (D898N) and TET2 (T1114S) were found in the cribriform ACC component. In fact, the PIK3C2G (R781C), PTCH1 (D898N) and TET2 (T1114S) mutations may be the founder genetic events, as they were present in both the cribriform and the solid components with the cancer cell fraction ranging from 48% to 52%, whereas the KDM6A (splice site 564+1G>C) mutation was restricted to only the cribriform component. However, four mutations targeting 3 genes that affect bona fide cancer genes, FGFR2 (Y375C), NOTCH1 (A2478fs, A1742fs) and CREBBP (E1000*), were verified to be restricted to solid component (Figure 2B ,2C and 2D).
In 2 of the 5 cases (ACC4813 and ACC4802), the MYB-NFIB fusion gene involving MYB exon 14 and NFIB exon 9 was detected in both the cribriform/tubular and solid components, suggesting that MYB-NFIB fusion was an early event in the tumorigenesis of primary ACC.
Intertumor (primary/recurrent/metastasis) heterogeneity of solid type ACCs
Solid type ACC is known to be related to poor prognosis, local relapse, and distant metastasis. To characterize the genetic evolution that drives its propensity for recurrence or metastasis of the solid type, two aggressive ACCs were included in our study. These two cases were both diagnosed as solid type ACC with infiltrative borders in the primary tumors. Patient 4820 (ACC 4820) and Patient 1958 (ACC 1958) showed local recurrence at 11 and 16 months after surgery respectively. Distant metastases to the lung were clinically noted in Patient 1958 at 13 months after surgery and then proven by biopsy. We performed NGS on the primary and paired recurrent tumor specimens of both patients and the distinct lung metastases of Patient 1958.
In ACC 1958, the MYB-NFIB fusion gene and 9 somatic mutations were all detected in the primary, recurrent and metastatic tumor specimens. However, one missense mutation and one frameshift mutation in NOTCH1 (L1709P, S2467fs) were both significantly enriched in the metastatic specimen with cancer cell fractions of 45-46% in primary ACC, 45-48% in local reoccurring ACC vs. 65-68% in metastatic ACC, suggesting that NOTCH1 may be a driver gene in the progression of ACC distant metastasis. In addition, a subclonal mutation of SPEN (R1475*), a member of the NOTCH pathway as a known histone modification gene, was found in the metastatic specimen, further supporting the role of the NOTCH pathway in ACC (Figure 2D and 2E).
In ACC 4820, mutations in AR and CHEK2 were identified as the founder genetic event, being present and clonal in both the primary and recurrent specimens with cancer cell fraction of more than 90%. Akin to ACC1958, NOTCH1 mutation was also detected in both the primary and recurrent specimen, although with no enrichment in the cancer cell fraction. In addition, chromosome 1, the area in which the NOTCH2 and HSD3B1 genes are located, showed ampliﬁcation restricted to the local recurring specimen (Figure 2D and 2E, Supplementary Figure 2).
From the above, we concluded that primary, recurrent and metastatic specimens shared consistent main alterations. SPEN, a member associated with the NOTCH pathway, is not only important in the tumorigenesis of solid type ACC, but also plays an early and/or an initiating tumorigenic role in primary tumor development and metastatic progression. In addition, the amplification of NOTCH2 and HSD3B1 on chromosome 1 may promote the local relapse of solid type ACC.
Immunohistochemical characteristics in the different components of the solid type ACCs
To identify the pathologic features of distinct components (cribriform/tubular and solid) within a solid type ACC, an immunohistochemical study of the myoepithelial marks of p63 and p40, together with the luminal markers CK7, and Ki-67 was carried out. The solid component was diffusely positive for CK7 ( Figure 3A, d and f) and negative or weak for p63 (Figure 3A, g and i) and p40 (Figure 3A, j and l) with the loss of the biphasic appearance, while CK7 (Figure 3A, d and e) and p63/p40 (Figure 3A, j and k) highlighted the epithelial and myoepithelial cells respectively in the lesions of conventional ACC. Ki-67 immunostaining indicated that the proportion of proliferating cells was more than 10% (positive nuclear staining) in the solid component and <5% in the cribriform/tubular component (Figure 3A, m, n and o). The same was true of p63/p40 and CK7 immunostainings in the solid pattern of the other 26 solid type ACCs (Supplementary Figure 3). These results indicated that gland differentiation with the absence of myoepithelial cell differentiation was a major feature of solid type ACCs.
FISH analysis in the different components of the solid type ACCs
To confirm the gene rearrangement results from NGS in 31 solid type ACCs, we investigated MYB status by means of FISH. In the 7 cases that NGS showed MYB-NFIB/APOO fusion, FISH analysis displayed two kinds of patterns: one was the typical pattern with an intact signal (fused orange/green signals) and a split signal (separated orange and green signals), indicating a breakpoint within the MYB gene (4/7) , and the other was an atypical pattern with one fusion signal and one green signal, indicating the loss of the 3′ end (3/7). In the case NGS presented MYB truncation (case 4819), the atypical pattern with one fusion signal and one green signal was observed (Data not shown). Intriguingly, in ACC 4813 and ACC 4802 which arranged in distinct growth patterns, the typical patterns with an intact signal and a split signal were shown in the cribriform components (Figure 3B-b and Supplementary Figure 4b), while the atypical pattern with one fusion signal and one green signal, indicating the loss of the 3′ end (2/5), was present in the solid component (Figure 3B-a and Supplementary Figure 4a).
NOTCH1 mutation and NCID expression
The correlation between NOTCH1 mutation and prognosis was analyzed in solid type ACCs. Kaplan–Meier analyses showed that patients with NOTCH1/NOTCH2 alteration were inclined to have lower distant metastasis-free survival rates (P=0.032) than those with no NOTCH1/NOTCH2 mutation, but there was no difference in the overall survival and recurrence-free survival between the patients in the two groups (Figure. 4A). The association between NOTCH1 mutations and NOTCH1 activation was then determined. The immunostaining of NCID, an established marker of NOTCH1 activation was performed. A statistically significant association between NOTCH1 mutations and NICD positivity was identified (Figure 4B). The H score of nuclear NICD1 expression in 20 tumors with NOTCH1 potentially activating mutations was much higher than that in 11 tumors with NOTCH1 wild-type or predicted NOTCH1 mutation to be inactivating (190.5 ± 13.05 vs 71.8 ± 9.61, P＜0.0001, Figure 4C). There was no difference in NICD1 expression between tumors with one and multi-hit NOTCH1 mutations predicted to be activating (165.7 ± 16.88, N=7 vs 203.8 ± 17.19, N=13, Figure 4D).
The genetic profiles of ACC with high-grade transformation
Recently, several studies reported that a distinct entity of ACC with high-grade transformation (HGT) often gradually transforms from solid type ACC, but other than p53 mutation21, the molecular genetic mechanisms responsible for these transformations remain largely unknown. Therefore, our study included 5 cases of ACC with HGT to concentrate on genetic alterations (Table 3). The mean age of the five patients was 47.4 years (range, 20-64 years), and all patients were male. The mean size of the tumor was 4.1 cm (range, 1.9-5.5 cm). In all cases, the transformed components comprised more than 30% of the entire lesion and displayed larger and more pleomorphic, vesicular nuclei. Micropapillary growth was noted and necrosis was common (Figure 5B-a, b and Figure 5C-a, b). Neck dissections were performed in addition to surgical excision in 3 cases in which imaging tests and subsequent pathological slices indicated lymph node metastasis.
The high-grade transformation component was the target lesion that was microdissected for NGS. MYB-NFIB fusion existed in only one case (Figure 5A). However, the multi-hit mutations of NOTCH1 predicted to be activating were observed in 3 cases and a NOTCH1 rearrangement of unclear significance was found in another case. Since NOTCH1 mutations are fundamental and critical in the tumorigenesis and progression of solid type ACC, our results proposed that ACC with HGT was not an abrupt transition from low-grade ACC but may be a gradual transformation from solid type ACC. Most importantly, somatic mutations of genes including PIK3CA, PIK3R1 and PPP2R2A associated with the PI3K/AKT pathway were identified in 4 cases of ACCs with HGT. PIK3CA encodes the PI3K catalytic subunit p110α, and PIK3R1 encodes the PI3K regulatory subunit p85α, and PPP2R2A is a potent negative regulator of AKT. Mutations in PIK3CA occurred in the helical domain (E545K) and the kinase domain (H1047R) and mutations in PIK3R1 that occurred in iSH2 domains (N564D) were all hotspots for the activation of the canonical PI3K pathway. In the remaining case, p53 mutation and FGFR2 amplification played an important role in the progression to high-grade transformation. Above all, we concluded that the gain of function mutations in p53, PI3K/AKT related genes or FGFR2 amplification were essential events in the progression from solid type ACC to HGT ACC.
As described above, the expression levels including CK7, p63, NCID, the PI3 Kinase p110α, p53 and pAKTT308 were analyzed. P63 immunostaining was negative and CK7 immunostaining was weakly or moderately positive in the high-transformation grade area, indicating loss of the biphasic appearance. Ki-67 immunostaining was more than 50% positive, indicating a high proliferation ability. The cases with NOTCH1 mutations showed high expression of NCID, while that with NOTCH1 rearrangement presented negative expression of NCID. Strong and diffuse expression of p53 was observed in Patient 4821. Of the 4 tumors with mutations in PI3K/AKT related genes, positivity was observed for p110α and pAKTT308 with a high intensity and extent of staining (Figure 5B and 5C).
The in vivo evaluation of NOTCH1 inhibitor BMS-906024 in high grade ACC PDXs
A panel of ACC PDXs were established by our group. PDX 34 was from ACC 4820 which harbored a multi-hit mutation of NOTCH1. The HE staining and IHC staining of PDX 34 was done to confirm the histological consistency to ACC 4820. The xenograft tumor exhibited solid type and diffusely positive for CK7 and negative for p63. As well, it showed a high expression of NCID indicating the activation of NOTCH signaling pathway. As NOTCH1 is the most frequently mutated gene in high grade ACCs, a preclinical evaluation of γ-secretase inhibitor (GSI) BMS-906024 inhibiting NOTCH signaling was conducted on PDX 34. Single-agent treatment with BMS-906024 (8.5 mg/kg) or cisplatin (5 mg/kg) were performed in the PDX models for 28 days. BMS-906024(TGI=48%) reduced the volume and weight of xenograft tumors and exhibited a better tumor growth inhibitory effect than cisplatin (TGI=12%). In addition, the toxicities of BMS-906024 and cisplatin were well tolerated during the treatment period. To confirm that the TGI induced by BMS-906024 was a consequence of a downregulation of NOTCH1, we performed IHC staining of NCID in the PDX tumors after the treatment. Treatment of PDX 34 with BMS-906024 inhibited NCID expression much better than cisplatin and the control. However, cell proliferation, which assessed by Ki67 staining, was reduced to varying degrees in cisplatin and BMS-906024 groups.